The Role of N-terminal domain of RAD54 in Branch Migration of Holliday Junctions
By
Nadish Goyal
April 2017
A Dissertation
Submitted to the Faculty
of
Drexel University College of Medicine
in partial fulfillment of the
Requirements for the Degree
of
Doctor of Philosophy
April 2017
iii
ACKNOWLEDGEMENTS
I would like to thank my mentor, Dr Alexander Mazin, for allowing me to work in his lab and for his patience, belief and untiring support throughout my tenure as a graduate student. It has truly been a pleasure having insightful discussions leading to exciting research with such an enthusiastic advisor. I would also like to express my deepest gratitude to Dr. Olga Mazina for her immense support and assistance on various experimental techniques, and for the all the conversations we have had that made my experience a memorable one. I would like to thank the members of my thesis advisory committee, Dr. Simon
Cocklin, Dr. Patrick Loll, Dr. Paul McGonigle, Dr. Eishi Noguchi, and Dr. Christian
Sell, for their time, encouragement, and thoughtful inputs given to me during the preparation of my dissertation.
I would also like to thank my friends, current and previous members of the
Mazin lab, all of whom have been extremely generous with their time and knowledge. I would also like to extend my gratitude to fellow colleagues who provided help during my coursework.
Finally, I would like to thank my family, especially my parents, for all their love, support, and encouragement showered upon me throughout the course of my academic career. Thank you for all the love, support, patience, and always willing to do absolutely anything you could to help me reach my goals. I am very grateful to them for the work ethic that they instilled in me. It is by their example that I continued to put in consistent and persistent efforts. I would also like to
iv thank my sister for providing all the help whenever needed. I would also like to thank all of the people who have helped me over the past six years.
v
TABLE OF CONTENTS
LIST OF TABLES ...... ix
LIST OF FIGURES ...... x
ABSTRACT ...... xiii
CHAPTER
1. Introduction ...... 1
1.1 Multi-protein complexes involved in Eukaryotic Homologous recombination repair ...... 4
1.1.1 Proteins involved in pre-synaptic resection of double-strand break
ends ...... 5
1.1.2 Role of RAD51 recombinase and RAD54 helicase-like protein during
Homologous Recombination ...... 7
1.2 Functions of RAD54 in homologous recombination ...... 10
1.3 Structural features of RAD54 protein ...... 13
1.4 Biochemical activities of RAD54 protein ...... 18
1.4.1 RAD54 has ds-DNA dependent ATPase activity ...... 18
1.4.2 RAD54 translocates on ds-DNA...... 18
1.4.3 RAD54 promotes chromatin remodeling ...... 20
1.4.4 Functional interaction between RAD54 and RAD51 ...... 22
vi
1.4.5 RAD54 promotes branch migration of Holliday junctions ...... 25
1.5 Role of RAD54 branch migration activity in homologous recombination ...... 31
1.6 Resolution of Holliday junction ...... 33
1.7 Regulation of RAD54 activities ...... 35
1.8 Protein regulation based on its oligomeric state ...... 37
1.9 Small-molecule inhibitors of homologous recombination proteins ...... 38
1.10 Summary ...... 41
2. RAD54 N-terminal domain: a DNA sensor that couples ATP hydrolysis with branch migration of Holliday junctions ...... 44
2.1 Abstract ...... 44
2.2 Introduction ...... 45
2.3 Materials and Methods ...... 48
2.4 Results...... 58
2.4.1 The RAD54 N-terminal domain is essential for branch migration of
Holliday junctions ...... 58
2.4.2 RAD5496-747 retains DNA translocation activity ...... 62
2.4.3 The N-terminal 95 aa truncation changes DNA binding preferences
of RAD54 ...... 66
2.4.4 The RAD54 N-terminal domain binds preferentially to branched DNA
substrates ...... 67
vii
2.4.5 Mutations that separately inhibit N-terminal domain oligomerization
or DNA binding ...... 69
2.4.6 The N-terminal domain mutations inhibit RAD54 branch migration .... 74
2.4.7 N-terminal domain phosphorylation by CDK2 inhibits RAD54 branch
migration but not its stimulation of RAD51 strand exchange ...... 77
2.5 Discussion ...... 82
3. Characterization of the RAD54 promoted annealing activity ...... 89
3.1 Introduction ...... 89
3.2 Materials and Methods ...... 93
3.3 Results...... 97
3.3.1 RAD54 promotes annealing of complementary ssDNA strands ...... 97
3.3.2 Effect of nucleotide cofactors on RAD54 promoted annealing ...... 98
3.3.3 N-terminal domain is necessary but not sufficient for annealing
activity of RAD54 ...... 100
3.3.4 DNA binding by N-terminal domain, not oligomerization, is required
for ssDNA annealing promoted by RAD54 ...... 101
3.3.5 RAD54 promotes annealing of ssDNA and ssRNA strands ...... 103
3.4 Discussion ...... 104
viii
4. Identification of small-molecule inhibitors of RAD54 ...... 107
4.1 Introduction ...... 107
4.2 Materials and Methods ...... 109
4.3 Results...... 113
4.3.1 High-throughput screening for RAD54 inhibitors ...... 113
4.3.2 Validation of hits identified by high-throughput screening ...... 114
4.3.3 Analysis of compounds in DT-40 chicken cells ...... 117
4.3.4 Screening of second generation inhibitors of RAD54...... 119
4.4 Discussion ...... 122
5. Conclusions and future directions ...... 125
6. Bibliography ...... 137
Appendix A Targeting BRCA1- and BRCA2- deficient cells with small- molecule inhibitors of RAD52 ...... 161
A.1 Abstract ...... 161
A.2 Introduction ...... 162
A.3 Materials and Methods ...... 165
A.4 Results ...... 176
A.4.1 High-throughput screening for RAD52 inhibitors ...... 176
ix
A.4.2 Analysis of RAD52 inhibitors in human BRCA1- and BRCA2-
deficient cells ...... 185
A.4.3 Measurement of inhibitors binding to RAD52 by SPR ...... 190
A.4.4 Inhibitors disrupt RAD52, not RAD51, foci formation ...... 192
A.4.5 D-I03 inhibits single-strand DNA annealing, but not gene conversion
in U2OS cells ...... 195
A.5 Discussion ...... 201
x
LIST OF TABLES
Table 2.1 Sequences of oligonucleotides used in chapter 2 ...... 48
Table 2.2 Sequences of primers used in chapter 2 ...... 52
Table 3.1 Sequences of oligonucleotides used in chapter 3 ...... 93
Table 4.1 Sequences of oligonucleotides used in chapter 4 ...... 110
Table 4.2 IC50 values for compounds in specificity assay ...... 116
xi
LIST OF FIGURES
Figure 1.1 The DNA double-strand break repair pathways by homologous recombination ...... 2
Figure 1.2 Strand exchange activity of RAD51 ...... 8
Figure 1.3 Structure of RAD54 protein ...... 16
Figure 1.4 Sequence alignment of N-terminal domain of RAD54 orthologs ...... 17
Figure 1.5 RAD54 translocates on ds-DNA ...... 19
Figure 1.6 RAD54 promotes chromatin remodeling ...... 21
Figure 1.7 RAD54 stimulates strand exchange activity of RAD51 ...... 23
Figure 1.8 RAD54 promotes branch migration of Holliday junctions ...... 26
Figure 1.9 RAD54 stimulates endonuclease activity of Mus81 ...... 34
Figure 2.1The RAD54 N-terminal domain is essential for branch migration, but not for ATP hydrolysis ...... 62
Figure 2.2 The 95 aa truncation enhances DNA translocation activity of RAD54 .. 66
Figure 2.3 The DNA binding preference of RAD541-142 ...... 69
Figure 2.4 Mutations in RAD541-142 disrupt its DNA binding ...... 73
Figure 2.5 The S49E, but not RKRK33-36AAAA, mutation inhibits DNA- dependent oligomerization of RAD541-142 ...... 74
Figure 2.6 Comparison of activities of RAD54 purified from E. coli or insect cells . 75
Figure 2.7 The S49E and RKRK33-36AAAA mutations inhibit branch migration, but not ATP hydrolysis ...... 77
xii
Figure 2.8 CDK2 phosphorylates N-terminal region of RAD54 ...... 79
Figure 2.9 Effect of CDK2 phosphorylation on branch migration and ATPase activities of RAD54 ...... 80
Figure 2.10 Effect of phosphorylated RAD54 on strand exchange activity of
RAD51 ...... 82
Figure 2.11 Model for CDK2-dependent RAD54 regulation ...... 87
Figure 3.1 RAD54 promotes ssDNA annealing...... 98
Figure 3.2 Effect of nucleotide cofactor on RAD54 annealing activity ...... 99
Figure 3.3 N-terminal domain is necessary but not sufficient for annealing activity of RAD54 ...... 101
Figure 3.4 DNA binding by N-terminal domain of RAD54 is necessary for annealing activity ...... 103
Figure 3.5 RAD54 promotes annealing of ssRNA and ssDNA ...... 104
Figure 4.1 Fluorescence-based assay for identification of RAD54 small-molecule inhibitors ...... 114
Figure 4.2 Validation of 55 RAD54 inhibitors using branch migration assay ...... 115
Figure 4.3 Effect of RAD54 inhibitors on survival of RAD18-deficient and wild type DT-40 cells ...... 118
Figure 4.4 Structures of first generation RAD54 inhibitors ...... 119
Figure 4.5 Screening for 32 second generation inhibitors of RAD54 ...... 120
Figure 4.6 Structures of second generation RAD54 inhibitors ...... 121
xiii
ABSTRACT
The role of RAD54 N-terminal domain in branch migration of Holliday
junctions
Nadish Goyal
Alexander V. Mazin, Ph.D.
Homologous recombination plays an important role in repairing the most harmful types of DNA damage, including DNA double-strand breaks and inter- strand cross-links, in promoting faithful chromosome segregation during meiosis, and in telomere maintenance. RAD54 protein, a member of theSwi2/Snf2 family of ATP-dependent DNA translocases, is crucial to the homologous recombination pathway. The protein is conserved in all eukaryotes. RAD54 is a multifunctional protein that promotes branch migration of Holliday Junctions, chromatin remodeling, and stimulation of DNA strand exchange activity of RAD51. The structure of RAD54 lacking the N-terminal domain has been solved. The central domain of RAD54 has DNA dependent ATPase motifs that are well conserved in all members of helicase Superfamily 1 and 2; however, the N-terminal domain is unique for RAD54 orthologues and is not well understood.
Here, using biochemical approaches and site-directed mutagenesis, we characterized the role of the RAD54 N-terminal domain in branch migration of
Holliday junctions. We found that by removing N-terminal domain residues we were able to uncouple ATPase and branch migration activities of RAD54. Also,
xiv we found that a recently identified DNA binding site in the N-terminal domain of
RAD54 has a preference for branched DNA substrates similar to that of the full length RAD54. Disruption of DNA binding of the N-terminal domain inhibited the branch migration activity of RAD54 without affecting its ATPase activity. Our results demonstrate for the first time that the DNA binding by the N-terminal domain is necessary for the branch migration activity of RAD54. This requirement of second DNA binding domain for branch migration may be conserved among branch migration proteins.
We also identified specific small-molecule inhibitors for branch migration activity of RAD54. Interestingly, the initial analysis showed that two small- molecule compounds, C-A23, and C-G01 had a stronger inhibition on branch migration without significantly affecting its ATPase activity. This suggests that they may inhibit branch migration by interacting with the N-terminal domain of
RAD54. However, further work needs to done to determine their exact mechanism of action and the function of branch migration in cells using these inhibitors.
1
INTRODUCTION
Homologous recombination is a highly conserved mechanism involved in the maintenance of genomic stability by repairing toxic DNA lesions such as double-strand breaks, DNA inter-strand crosslinks and stalled replication forks in somatic cells. Homologous recombination pathway is also required for accurate segregation of chromosomes during meiosis by generating crossovers between paternal and maternal chromosomes (Bishop, 2006; Neale and Keeney, 2006;
Pâques and Haber, 1999; Stark and Jasin, 2003). All homologous recombination pathways are generally considered error-free as they require an intact homologous strand of DNA to serve as a donor template for repair of the broken chromosome.
The process of homologous recombination involves recognition of a double-strand
break site, resection of DNA ends with the help of endonucleases to generate DNA
duplex with protruding 3’ single-stranded DNA tails (Figure 1.1). RAD51
recombinase binds to the resected ssDNA tails to form a nucleoprotein filament
that searches for homologous double-strand DNA. Once homology is found, the
nucleoprotein filament invades the homologous duplex DNA which results in the
formation of displacement loop or D-Loop. After the D-loop intermediate is formed,
3’-end of the invading strand is extended followed by capture of the second strand of the broken chromosome by a process called strand annealing, finally resulting in the formation of double D-loops that are converted to Holliday junctions. Branch migration and resolution of Holliday junctions ultimately result in generating crossover or non-crossover products (Gravel et al., 2008; San Filippo et al., 2008;
Venkitaraman, 2002).
2
Figure 1.1 The DNA double‐strand break repair by Homologous Recombination. The initial steps involve, 5’ to 3’ exonucleolytic processing of double-strand break ends to produce 3’‐ssDNA tails, the formation of Rad51‐ ssDNA filaments, search for homology and strand invasion into the homologous duplex DNA‐template leading to the formation of displacement loops (D‐loop). Then, homologous recombination may proceed either by (A) SDSA forming non‐ crossover products or (B) DSBR forming crossover products.
3
In addition to the Homologous Recombination pathway, DNA double-strand breaks can also be by Non-Homologous End-Joining. The selection of the mechanism of double-strand break repair depends on whether the DNA ends at the double-strand break site are resected or not. While the Non-homologous End joining pathway is effective only when the double-strand break ends are intact and not resected (Mimitou and Symington, 2008), the homologous recombination pathway requires the resected 3’ ssDNA tail to proceed as mentioned above
(Figure 1.1). Hence, resection of double-strand break ends can be termed as the committed step in the homologous recombination pathway. This step is accomplished by specialized multifunctional enzyme complexes that contain DNA break recognition proteins, helicases, and nucleases that are regulated by specific
DNA sequences in prokaryotes and cyclin-dependent kinases in eukaryotes.
Various components of the homologous recombination pathway initiate the repair process upon resection of DNA ends at the double-strand break site. Depending on the mode of DNA repair, homologous recombination is sub-categorized into two major pathways namely, double strand break repair pathway, DSBR, and
synthesis dependent strand annealing pathway, SDSA (Figure 1.1). The DSBR
pathway is characterized by the formation of a double-Holliday junction, unlike the
SDSA pathway which involves the formation of a single Holliday junction. In the
SDSA pathway, the D-loop complex formed by the extension of the invading strand by DNA polymerase on the homologous DNA sequence dissociates by branch migration and anneals with the complimentary resected end as opposed to the
DSBR pathway in which second-end capture takes place. Hence, SDSA pathway
4 produces only non-crossover products as a result of bypassing the second-end capture step (Nimonkar et al., 2008; Zhu et al., 2008). This pathway is especially important during DNA double-strand break repair in somatic cells. In contrast, the
DSBR pathway that generates crossing over is essential for segregation of homologous chromosomes during meiosis (Allers and Lichten, 2001).
1.1 MULTI-PROTEIN COMPLEXES INVOLVED IN EUKARYOTIC HOMOLOGOUS RECOMBINATION REPAIR
Early studies to understand the mechanism of homologous recombination
in eukaryotes were carried out in yeast. In particular, Saccharomyces cerevisiae
was used as a model organism for studying homologous recombination. Several
key proteins were identified in S. cerevisiae mutant screens which demonstrated
increased sensitivity to ionizing radiation with little sensitivity to ultraviolet radiation
(Game and Mortimer, 1974). These proteins which included Rad50, Rad51,
Rad52, Rad54, Rad55, Rad57, Rad59, Mre11, and Xrs2 belongs to the RAD52
epistasis group that constitutes the core of the homologous recombination
enzymatic machinery (Sommer et al., 1998; Story and Steitz, 1992). Each of these
proteins has a distinct role to play in both mitotic and meiotic recombination.
Furthermore, a few members of the Rad52 epistasis group such as Rad50, Rad51,
Rad52, Rad54 and Mre11 have mammalian analogues which make them critical
proteins in the homologous recombination pathway.
5
1.1.1 Proteins involved in pre-synaptic resection of double-strand break
ends
In yeast, a heterotrimeric protein complex known as the MRX (Mre11-
Rad50-Xrs2) complex participates in processing of the DNA double-strand break
ends to generate 3’-ssDNA tails (Figure 1.1). In higher eukaryotes, including
humans, its homologue is known as MRN (MRE11-RAD50-NBS1). The end
resection occurs in two steps in yeast and follows the same pattern in humans as
well, which involves initial limited resection followed by a processive resection
(Neveling et al., 2009). The MRX or MRN complex participates only in the initial
limited resection. Of these proteins, Mre11 is a highly conserved eukaryotic
nuclease protein that binds first to the DNA double-strand break ends (Lindsley
and Cox, 1990). As with most other yeast proteins, Mre11 is the yeast homolog of
human MRE11. Mre11 initially forms a complex with Rad50 and is recruited to the
double-strand break site for nucleolytic processing. Another yeast protein Xrs2,
which is also a functional homolog of human Nbs1, associates with Mre11 and
Rad50 to form the heterotrimeric Mre11/Rad50/Xrs2, MRX complex in yeast. The
function of Xrs2 in yeast or Nbs1 in humans has not been fully understood but
several studies demonstrate Xrs2 to be involved in targeting Mre11 and Rad50 to
the double-strand break site. Once formed, the MRX complex further associates
with the Sae2 nuclease to eliminate a short nucleotide from double-strand break
ends to complete the initial limited resection (Lieber et al., 2003). Protein CtIP is
the human homolog of yeast Sae2 protein and shares a region of homology with
Sae2 in its C-terminal domain containing 108 amino acids. CtIP protein performs
6 a similar function of stimulating Mre11 endonucleolytic activity (Neveling et al.,
2009; West et al., 1998). Several studies demonstrated that yeast strains carrying mutations in Mre11, Rad50 or Xrs2 genes showed defects in meiotic recombination and DNA repair. These mutants also show delayed processing of double-strand break ends in mitotic cells thereby confirming the importance of the
MRX complex in early stages of homologous recombination (Galletto et al., 2006;
Lindsley and Cox, 1990; McEntee et al., 1981; Sattin and Goh, 2004).
Following the initial limited resection, two distinct pathways emerge for the processive resection (Neveling et al., 2009). One pathway employs a 5’ to 3’ exonuclease protein, Exo1, whereas the other pathway utilizes two proteins – Sgs1 helicase and Dna2 nuclease. The human homologs for several of the yeast proteins involved in the end-resection pathway have been identified. The human homolog of yeast Sgs1 is BLM helicase, and both the proteins belong to the RecQ family of helicases. Also, yeast Exo1 and human Exo1 protein sequences are 27% identical and 55% similar suggesting comparable functional properties (Walker et al., 2001). In vitro studies demonstrated that BLM stimulates human Exo1 resection activity by physically interacting with it in an ATP independent manner
(Walker et al., 2001). This complex assembly of proteins exemplifies the importance of DNA double-strand break end resection in homologous recombination.
7
1.1.2 Role of Rad51 recombinase and Rad54 helicase-like protein during
Homologous Recombination
Once the double-strand break ends undergo resection to produce 3’-ssDNA
overhangs, Rad51 recombinase binds to ssDNA ends, forming a contiguous nucleoprotein filament (Kowalczykowski, 2008). This nucleoprotein filament then searches for homologous DNA sequence to use as a template for double-strand break repair (Chen et al., 2008; Kowalczykowski, 2008; Ogawa et al., 1993;
Stasiak and Di Capua, 1982). One model for homology search comprises cycles of binding and releasing potential homologous templates until homology is located.
This is the random collision model and is ATPase-independent. (Kowalczykowski,
1991; Sung et al., 2003). Another model, sliding model wherein the nucleoprotein filament can diffuse along the dsDNA track has recently gained an experimental support (Ragunathan et al., 2012). Once homology is found, the nucleoprotein filament invades the homologous DNA template, displacing one of the DNA
strands of the duplex DNA, forming a displacement loop (D-loop or joint molecule)
(Figure 1.2). The invading DNA strand is then extended by DNA polymerase and the cross structure formed at the point of strand exchange is known as a Holliday junction (Holliday, 2007; Liu and West, 2004). Depending upon the DNA repair pathway, the Holliday junction can either be dissolved or extended by movement along the DNA axis in a process called branch migration. Several proteins were identified in eukaryotes able to perform branch migration of Holliday junctions;
RAD54 is the most potent among them (Mazin et al., 2010). The structure-specific
8 endonuclease, Mus81-Eme1, can carry out the resolution of the Holliday junctions to produce crossover or non-crossover products and complete the repair process
(Heyer, 2004; Liu and West, 2004).
Figure 1.2 Strand-Exchange activity of RAD51. The in vivo strand-exchange activity of RAD51 (left) is mimicked using a D-loop formation assay in vitro (right). The nucleoprotein filament is formed by incubation of RAD51 protein with single- stranded DNA, following which homologous double-stranded DNA (pUC19) is added to form D-loops.
Rad51, a member of the RAD52 epistasis group, is a highly conserved recombinase. Rad51 shares structural homology with RecA recombinase (30% identity) found in E. coli (Sung et al., 2003). Rad51 binds single-stranded DNA forming a right handed helical filament, in which DNA structure is significantly extended (Story et al., 1992; Sung et al., 2003). Rad51/RecA has two DNA binding
9 sites: the primary binding site is involved in filament formation and the secondary binding site allows for the capture of duplex DNA during the search for homology
(Kowalczykowski, 1991; Sung et al., 2003). Rad51’s ability to differentiate between single-stranded and double-stranded DNA as well as the stability of the Rad51- single-stranded DNA filament can be modulated by different ions (Liu et al., 2004;
Shim et al., 2006; Sigurdsson et al., 2001). In presence of Ca2+ ion, hRad51 forms
an active and stable Rad51-single-stranded-DNA filament whereas, in presence
of Mg2+ ion, it forms an inactive filament (Bugreev and Mazin, 2004). Ca2+ slows
down the rate of ATP hydrolysis by Rad51, preventing Rad51 dissociation from
nucleoprotein filament, and thus stimulates strand exchange and joint molecule
formation between single-stranded DNA and the homologous duplex DNA
(Bugreev and Mazin, 2004). This Ca2+ dependent stimulation may be biologically
relevant because an elevation in Ca2+ levels has been observed in human cells
during the DNA damage response (Gafter et al., 1997; Negre-Salvayre and
Salvayre, 1992; Sakai et al., 1994; Schieven et al., 1993; Spielberg et al., 1991)
as well as during meiosis I when homologous recombination is thought to take place (Carroll et al., 1994; Tombes et al., 1992). Therefore, by modulating Rad51’s
ATPase activity, filament stabilization and strand exchange activity of Rad51 can
be regulated (Bugreev and Mazin, 2004).
Rad54 has been shown to stabilize Rad51 nucleoprotein filaments (Mazin
et al., 2003). Rad54 interacts physically and functionally with Rad51 (Clever et al.,
1997; Raschle et al., 2004), and stimulates the strand exchange activity of Rad51.
10
Rad54 belongs to the SWI2/SNF2 family of helicase-like proteins and is evolutionarily conserved. The members of this family of helicase-like proteins are known for their chromatin remodeling activity. The helicase-like proteins do not have DNA strand separation activity like canonical helicases, however, they use
DNA-dependent ATP hydrolysis to translocate along the DNA generating positive supercoils in the DNA ahead and negative supercoils behind the protein. This allows the DNA strands to transiently “breathe”. Rad54 promotes chromatin remodeling and displacement of proteins from double-stranded DNA. Importantly, it binds Holliday junctions with high affinity and drives their branch migration in an
ATPase dependent manner (Bugreev et al., 2006b). Rad54 interacts with Mus81-
Eme1 (Mms4), a structure-specific endonuclease, stimulating its DNA cleavage activity (Matulova et al., 2009; Mazina and Mazin, 2008). Thus, Rad54 is a multifunctional protein performing functions throughout homologous recombination and linking the whole process together (Mazin et al., 2010).
1.2 FUNCTIONS OF RAD54 IN HOMOLOGOUS RECOMBINATION
The RAD54 gene was discovered in genetic screens for Saccharomyces cerevisiae mutants conferring strong sensitivity to ionizing radiation (IR), but only moderate sensitivity to ultra violet (UV) light (Game and Mortimer, 1974; Suslova,
1969; Zacharov, 1970). Complementation analysis identified it as a member of the
RAD52 epistasis group constituting the core of the homologous recombination enzymatic machinery (Symington, 2002). Rad54, along with Rad51 and Rad52, is
11 one of the three most IR-sensitive mutants in S. cerevisiae (Friedberg et al., 1995;
Game, 1993).
In vivo, Rad54 is a moderately abundant nuclear protein. There are an estimated 7× 103 and 2.4 × 105 Rad54 molecules per cell in exponentially growing
diploid yeast cells and in an unsynchronized population of mouse embryonic stem
(ES) cells, respectively (Clever et al., 1999; Essers et al., 2002a). In both yeast and mammals, Rad54 expression shows cell cycle dependence with an increase in transcription during late G1 phase (Cole and Mortimer, 1989; Cole et al., 1989;
Essers et al., 2002a; Johnston and Johnson, 1995). This increase in S and G2 phases is also observed with other recombination proteins indicating the important role of homologous recombination during DNA replication and may account for the increased resistance of cells to DNA double-strand break-inducing agents in G2
(Takata et al., 1998).
Rad54 is a well conserved protein of the Rad52 group, a generally well conserved group in eukaryotes. For instance, hRad54 shares 66% similarity and
48% identity with yRad54 (Kanaar et al., 1996; Petrini et al., 1997; Thoma et al.,
2005). Rad54 has homologues in other eukaryotes including S. pombe (Muris et
al., 1996), Arabidopsis (Shaked et al., 2006), Drosophila (Kooistra et al., 1997),
chicken (Bezzubova et al., 1997), zebrafish (Thoma et al., 2005), mouse, humans
(Haseltine and Kowalczykowski, 2009; Kanaar et al., 1996), and, at least, in one
archaean genus, Sulfolobus (Durr et al., 2005). Despite structural conservation
among the Rad52 group members, their functions in recombination and DNA
repair across different organisms are not fully conserved. In S.cerevisiae and S.
12 pombe, rad51 and rad54 mutants (rhp51 and rhp54, in S. pombe) show almost indistinguishable phenotypes with respect to the severity of their damage sensitivities (e.g., IR, UV-light), recombination, and chromosome loss in mitotic cells (Muris et al., 1993; Muris et al., 1996) whereas, the phenotypes of RAD51-/- and RAD54-/- mutants are significantly different in mice, as well as in chicken cells.
RAD51 gene disruption causes early embryonic lethality of mice. Even in cell
culture, the RAD51 knockouts fail to proliferate (Lim and Hasty, 1996; Sonoda et
al., 1998; Tsuzuki et al., 1996). In contrast, disruption of the RAD54 gene results in viable mice. However, the sensitivity of mouse embryonic stem cells and chicken
DT-40 cells to IR and methyl methanesulfonate (MMS) is increased (Bezzubova et al., 1997; Essers et al., 1997; Essers et al., 2000).
In mammals, the RAD54 gene plays the most important role during early developmental stages. In mice, RAD54-/- mutants show hypersensitivity to ionizing
radiation during early developmental stages which is rescued by Non-homologous
end joining pathway in adult mice (Essers et al., 2000). However, regardless of the
developmental stage, RAD54-/- mice are hypersensitive to DNA damage caused
by interstrand cross-linking agents (ICL), e.g., mitomycin C, which cannot be
repaired by the Non-homologous end joining (De Silva et al., 2000). Furthermore,
mutations in the RAD54 gene have been linked to breast, colon and lymphoma
cancer which is consistent with the role of Rad54 in maintaining genomic stability
(Matsuda et al., 1999).
Rad54 also plays an important, but less critical, role in other classes of
homologous recombination events. In S. cerevisiae, rad54 mutations cause a
13 relatively mild defect in mating-type switching, while rad51 and rad52 mutations completely abolish this recombination event (Schmuckli-Maurer and Heyer, 1999).
In S. cerevisiae, Rad54 plays a relatively minor role in meiotic recombination, due to the presence of a meiosis-specific homologue, Rdh54/Tid1 (Klein, 1997). The spore viability in the rad54 single mutant is reduced to 25-65% whereas the rad54 rdh54 double mutant is fully defective in viable spore formation, demonstrating that these two homologous proteins collectively possess the activities important for meiotic recombination (Schmuckli-Maurer and Heyer, 2000; Shinohara et al.,
1992; Shinohara et al., 1997). In mice, Rad54 plays a minor role in meiotic recombination, since RAD54-/- mutants are fertile (Essers et al., 1997). Meiosis-
specific Rad54 homologues are yet to be identified in higher eukaryotes.
Mammalian Rad54B, which was initially thought to be the Rad54 meiosis-specific
homolog, does not appear to have an important function in meiosis; as both
RAD54B-/- and RAD54-/- RAD54B-/- mice are fertile (Wesoly et al., 2006).
1.3 STRUCTURAL FEATURES OF RAD54 PROTEIN
Rad54 sequencing identified it as a member of the group of proteins
involved in translocation along nucleic acids that are broadly defined as helicases
(Gorbalenya and Koonin, 1993; Troelstra et al., 1992). The common feature of
these proteins is that they couple ATP hydrolysis to directional movement along
nucleic acids, which may or may not result in strand separation of nucleic acids.
Members of this group have a role in almost every cellular process from DNA
14 replication and repair, transcription, translation, splicing, and nuclear transport
(Singleton et al., 2007).
Rad54 is a member of Superfamily (SF) 2 of helicases (Troelstra et al.,
1992). Rad54 possesses seven classical “signature” motifs: I, Ia, II, III, IV, V, and
VI, a characteristic of SF2 protein members (Figure 1.3) (Nimonkar et al., 2007) plus several more recently identified motifs, like TxGx between motif Ia and motif
II that is thought to play a role in oligonucleotide binding. The seven conserved motifs line the two RecA-like motor domains, constituting the “core” or the minimal translocation motor responsible for the conversion of energy from ATP hydrolysis to the mechanical energy needed for translocation (Singleton et al., 2007).
Within SF2 superfamily, Rad54 belongs to the Snf2 (also known as
SWI2/SNF2) protein family of dsDNA-dependent ATPases. Unlike canonical DNA helicases, Snf2 family members cannot separate strands of the duplex DNA.
Instead, the Snf2 proteins can translocate along DNA, to carry out chromatin remodeling, DNA topology alterations, and displacement of proteins from DNA
(Haushalter and Kadonaga, 2003; Nimonkar et al., 2007). Several structural characteristics distinguish the Snf2 proteins from proteins of other SF2 families, e.g., DEAD-box RNA helicases, the RecQ helicase family, the DEAH helicase family, and others (Nimonkar et al., 2007). Each RecA-like lobe contains one of the Snf2-specific insertions HD1 and HD2 (helical domains 1 and 2). In the primary sequence, these protrusions are inserted between motifs III and IV, making the spacing between these two motifs much larger in Snf2 proteins as compared to the rest of the SF2 proteins (Flaus and Owen-Hughes, 2001).
15
X-ray crystallographic structures for two Rad54 orthologues, from zebrafish and Sulfolobus solfataricus, have been solved (Figure 1.3) (Durr et al., 2006; Durr et al., 2005; Thoma et al., 2005). The zebrafish Rad54 structure includes a part of the N-terminal domain, the two RecA-like α/β-domains (lobe 1 and 2) found in all
SF1 and SF2 helicases, and the carboxyl-terminal domain (Thoma et al., 2005).
Each RecA-like lobe contains one insertion HD1 and HD2, respectively, which appear as protrusions (Durr et al., 2005; Thoma et al., 2005). The carboxyl- terminal domain appears to be the only element unique to Rad54 that is present in the truncated structures. The carboxyl-terminal domain contains a tentative Zn- coordinating motif that may stabilize its entire assembly. The crystal structure of the catalytic domain of the S. solfataricus Rad54 homolog bound to a dsDNA substrate provided insight into the mechanism of Snf2 ATPases translocation along dsDNA, which is consistent with the inchworm translocation mechanism, similar to that described for other helicases (Singleton et al., 2007).
Though the RecA-like domains are conserved amongst the members of the helicase family proteins, the amino and the carboxyl-terminal domains of Rad54 are unique. Even among the Rad54 orthologues, the N-terminal domain is not well conserved (Figure 1.4). Proteolysis studies have shown that the N-terminal domain of Rad54 is relatively unstructured and was therefore removed from the structural studies (Raschle et al., 2004). A second DNA-binding site in the N-terminal domain of Rad54 has been reported recently (Wright and Heyer, 2014). The N-terminal domain of other DNA helicases has been shown to contain motifs that confer functional specificity to these proteins (Briggs et al., 2005; Soultanas et al., 2000).
16
Removal of the N-terminal domain in Saccharomyces cerevisiae Rad54 severely weakens its physical and functional interaction with Rad51 (Raschle et al., 2004).
The structures of the PcrA and RecG helicases show that the N-terminal domain is involved in protein-DNA interactions and allows these proteins to bind specifically to branched DNA structures (Singleton et al., 2001; Velankar et al.,
1999). Moreover, the structures of proteins from Superfamilies 3-6, show that these proteins form hexamers through amino-terminal contacts (Enemark and
Joshua-Tor, 2006; Huyton et al., 2003; Singleton et al., 2000; Skordalakes and
Berger, 2003). Therefore, the N-terminal domain of nucleic acid translocases might contain motifs that provide substrate specificity, contacts for protein-protein interactions, and the scaffold for oligomerization.
Figure 1.3 Structure of Rad54 protein. The X-ray crystal structure of Zebrafish Rad54 (PDB code: 1Z3I). The conserved Rec-A like motor domains are shown in blue and cyan. The N-terminal domain and C-terminal domain are unique to Rad54 orthologues and are shown in red and yellow, respectively.
17
S.cerevisiae 1 MARRRLPDR------PPNGIGAGE---RPRLV-PRPI---NVQDSV----NRLTK------PFRV 42 S.pombe 1 MIQQPTTAKPRISTSSKLNTVLSKNKENVPGKL-FKKF------KCP----SLVISEKRKELPLRK 55 Fruitfly 1 MRRSLAPSQ--R-GPLRPESR----HSFTPPLLKKNKRSCQQELEREQELDRRRLG------ALRD 53 Zebrafish 1 MRRSLAPSQ--V-AKRKQGPDSDDEEDWEPDMEPQSKR------DCR----EKYIS------PYRK 47 Chicken 1 L------AKRKAGGEE-EDGEWRPPAT-QKRQKAGSEAESA----DCYRS------PFRK 42 Mouse 1 MRRSLAPSQ--L-ARRKPEDRSSDDEDWQPGTVTPKKRKSSSETQVQ----ECFLS------PFRK 53 Human 1 MRRSLAPSQ--L-AKRKPEGRSCDDEDWQPGLVTPRKRKSSSETQIQ----ECFLS------PFRK 53
S.cerevisiae 43 PYK-NT--HIPPAAGRIATGSDNIVGGRSLRKRSATVCYSGLDINADEAEYNSQDI-SFSQLTKRR 104 S.pombe 56 KPRVNY--SEYG------SVDGKYDSAY-VSENVSGLATIKEAN 90 Fruitfly 54 A-SNTSELPLP------IR--FTANSEYEL------74 Zebrafish 48 PLTPLT--NRP------V---CADGNEHEA------66 Chicken 43 PLTQLT--NRP------L---CLDSSQHEA------61 Mouse 54 PLTQLL--NRP------P---CLDSSQHEA------72 Human 54 PLSQLT--NQP------P---CLDSSQHEA------72
S.cerevisiae 105 KDALSAQRLAKDPTRLSHIQYTLRRSFTVPIKG--YVQ-----RHSLPLTLGMKKKITPEPRPLHD 163 S.pombe 91 ------RLILNHER-RDPSTVIKKQFSVPKPIKGHEDISKLCAHRPPPTLGMKRKVDFIPRPLYD 148 Fruitfly 75 ------AIAKVLARKFKVPMDN--YVP-----DYGGKRVLGVRRC--ISRRPLHD 114 Zebrafish 67 ------FIRKILSKPFKIPIPN--YTG-----VL-GLRALGLRRA--GVRKALHD 105 Chicken 62 ------FIRSILSKPFKVPIPN--YKG-----PT-GLRALGIKRA--GLRSPLHD 100 Mouse 73 ------FIRSILSKPFKVPIPN--YQG-----PL-GSRALGLKRA--GVRRALHD 111 Human 73 ------FIRSILSKPFKVPIPN--YQG-----PL-GSRALGLKRA--GVRRALHD 111
S.cerevisiae 164 PTDEFAIVLYDPSVDGEMIVHDTSMDNKEEES-KKMIKSTQEKDN---INKEKNSQEERPTQRI-- 223 S.pombe 149 PADEFAIVLYDPTTDADEIIPDIKEVLAEKRKKDELLKNRKGKKEISDSEPESDHDSCVSTDTVAS 214 Fruitfly 115 PMACNALVLFHPP------127 Zebrafish 106 PFEDGALVLYEPP------118 Chicken 101 PFEEGALVLYEPP------113 Mouse 112 PLEEGALVLYEPP------124 Human 112 PLEKDALVLYEPP------124
S.cerevisiae 224 -GRHPALMTNGV-----RNKPLRELLGDSENSAENKKK--FASVPVVIDPKLAKILRPHQVEGVRF 281 S.pombe 215 CSTEQSLITSNTSKHRRPNKSLKDLLG------IQKEKPPPPPVAVVIDPKLARILRPHQIEGVKF 274 Fruitfly 128 ------AYTEHERMG------MDPTK---VLVHVVVDPLLSNILRPHQREGVRF 166 Zebrafish 119 ------AISAHDLIK------ADKEK---LPVHVVVDPVLSKVLRPHQREGVKF 157 Chicken 114 ------LLSAHEQLK------IDKDK---VPVHVVVDPVLSRVLRPHQREGVKF 152 Mouse 125 ------PLSAHDQLK------LDKEK---LPVHVVVDPVLSKVLRPHQREGVKF 163 Human 125 ------PLSAHDQLK------LDKEK---LPVHVVVDPILSKVLRPHQREGVKF 163
Figure 1.4 Sequence alignment of the N-terminal domain of Rad54 orthologs. The Rad54 aa sequences from Saccharomyces cerevisiae, Saccharomyces pombe, fruitfly (Drosophila melanogaster), zebrafish (Danio rerio), chicken (Gallus gallus), mouse (Mus musculus), and humans (Homo sapiens) were analyzed using multiple sequence alignment program, T-coffee (Di Tommaso et al., 2011). Pink, yellow, green, and blue colored regions show high, low, very low, and no conservation among sequences, respectively.
18
1.4 BIOCHEMICAL ACTIVITIES OF RAD54 PROTEIN
1.4.1 Rad54 has ds-DNA dependent ATPase activity
The biochemical activities of yeast and human Rad54 have been
extensively studied. Both the Rad54 orthologues were found to have robust
dsDNA-dependent ATP hydrolysis activity (Petukhova et al., 1998; Petukhova et
-1 al., 1999; Swagemakers et al., 1998), with a kcat ~ 3000-6000 min (Mazina et al.,
2007).
Rad54 binding to ss DNA does not stimulate its ATPase activity, although it
shows similar binding affinities for both ssDNA and dsDNA (Petukhova et al., 1998;
Swagemakers et al., 1998; Tanaka et al., 2002). Compared to linear ssDNA or
dsDNA, Rad54 shows a significantly higher binding affinity for branched DNA
structures. The binding preference is strongest, approximately 200-fold, for the
partial Holliday (PX) junction when compared to ssDNA or dsDNA fragments of the
same length (Bugreev et al., 2006b).
1.4.2 Rad54 translocates on ds-DNA
Unlike canonical helicases that have strand separation activity, Rad54 does
not show strand separation activity (Petukhova et al., 1998) but can translocate on
DNA. The ability of Rad54 to translocate on DNA was inferred from the biochemical
data; where it introduced equivalent positive and negative supercoiled domains
into closed circular dsDNA in an ATPase-dependent manner (Figure 1.5)
19
(Petukhova et al., 1999; Ristic et al., 2001; Tan et al., 1999; Van Komen et al.,
2000). In accord with Rad54’s ability to translocate on DNA, the rate of ATP hydrolysis increases with increase in the length of dsDNA (Mazina and Mazin,
2004). Also, as expected for DNA translocating protein, Rad54 is able to dissociate a DNA triple-helix (Jaskelioff et al., 2003) in the assay that was originally developed
to follow the translocation of a type I restriction endonuclease along DNA (Firman
and Szczelkun, 2000). The visual translocation of ScRad54 and ScRdh54 (Tid1),
a Rad54 meiosis-specific homologue using single-molecule analysis
demonstrated rapid and highly processive movement at ~300 bp/s for ScRad54
and 80-120 bp/s for ScRdh54 (Amitani et al., 2006; Nimonkar et al., 2007; Prasad
et al., 2007).
Figure 1.5 Rad54 translocates on ds-DNA. Rad54 (orange) binds to covalently closed circular plasmid DNA. The translocation of Rad54 (in the direction indicated by arrows), generates positive supercoils (+) ahead of the protein and negative supercoils (−) behind.
20
The precise structure of the active Rad54 complex that translocates on DNA remains unknown. However, it is believed that multimerization of the protein on
DNA helps to overcome the resistance to rotational motion of the protein around
DNA while generating supercoils in covalently closed DNA (Liu and Wang, 1987;
Ristic et al., 2001). Although both yeast and human Rad54 protein exist as a monomer in solution (Petukhova et al., 1999), Rad54 binding to DNA causes oligomerization of the protein (Petukhova et al., 1999); the cross-linking experiments indicated the formation of a dimer, as the principal oligomeric species, and larger oligomers (Mazina et al., 2007; Petukhova et al., 1999). Furthermore, the formation of oligomers by hRad54 and ScRad54 in complexes with DNA was also detected using atomic force microscopy (Ristic et al., 2001) and electron microscopy (Kiianitsa et al., 2006).
1.4.3 Rad54 promotes Chromatin Remodeling
Like other members of the Snf2 family, Rad54 promotes chromatin
remodeling, i.e., nucleosome redistribution along DNA, in an ATPase dependent
manner (Figure 1.6) (Alexeev et al., 2003; Alexiadis and Kadonaga, 2002;
Jaskelioff et al., 2003; Kwon et al., 2007; Wolner and Peterson, 2005; Zhang et al.,
2007). This chromatin remodeling activity may involve specific interactions of
Rad54 with histones in addition to its dsDNA translocation activity, as the N- terminus of ScRad54 interacts specifically with the N-terminal tail of histone H3
(Kwon et al., 2007). The chromatin remodeling activity of Rad54 may serve at least
21 two functions in homologous recombination: First, Rad54 could start clearing nucleosomes and other chromatin-bound proteins at the break site prior to end- processing. This is supported by the in vivo studies showing an effect of Rad54 on the accessibility of the HML donor site for the HO endonuclease (Wolner and
Peterson, 2005). Second, Rad54 might also facilitate the search for homology during synapsis by contending with chromatin structure of the target DNA. Indeed, in vitro studies show that Rad54 stimulates DNA strand exchange activity of Rad51 on chromatin-loaded DNA substrates to a greater extent than on naked DNA substrates (Alexiadis and Kadonaga, 2002). However, further work is required to fully understand the role of Rad54 chromatin remodeling activity in vivo.
Figure 1.6 RAD54 promotes chromatin remodeling. The chromatin remodeling makes DNA more accessible for RAD51 or other proteins, whereas, the protein displacement activity frees DNA for processing by other proteins.
22
1.4.4 Functional Interaction between Rad54 and Rad51
Rad54 functions in association with Rad51, a protein which promotes DNA strand exchange, a basic step of homologous recombination. Interactions between these two proteins are extensive and critical to the function of homologous recombination in eukaryotes (Clever et al., 1997; Krogh and Symington, 2004;
Rattray and Symington, 1995). Rad54 was found to interact functionally and physically with Rad51. To understand these interactions, extensive studies have been done over the last couple of decades. For example, in S. cerevisiae, Rad54 over-expression suppresses certain repair phenotypes of rad51 mutants (Clever et al., 1997; Morgan et al., 2002); in absence of Rad54, the rate and extent of
Rad51 recruitment to the HO-induced double-strand break is significantly reduced
(Wolner et al., 2003), co-localization of IR-induced Rad54 and Rad51 foci in
mouse ES cells (Tan et al., 1999). The physical interaction between Rad51 and
Rad54 proteins is species-specific and conserved from archea to humans (Clever et al., 1997; Golub et al., 1997; Haseltine and Kowalczykowski, 2009; Jiang et al.,
1996; Morgan et al., 2002). It has been previously reported that the Rad54 N-
terminal domain is primarily responsible for interactions with Rad51 (Alexiadis et al., 2004; Raschle et al., 2004). Rad54 can interact with free Rad51 protein and
also with Rad51-nucleoprotein filament, which is the active species in DNA strand
exchange (Mazin et al., 2003).
23
Figure 1.7 Rad54 stimulates strand exchange activity of Rad51. Rad54 interacts with free RAD51 and the active species during homology search, Rad51- ssDNA filament, and stimulates strand exchange activity of Rad51.
Rad54-dependent stimulation of Rad51’s DNA strand exchange activity
(Figure 1.7) was first observed for S. cerevisiae proteins but was later shown for archeal, Drosophila, and human orthologs as well (Alexiadis and Kadonaga, 2002;
Haseltine and Kowalczykowski, 2009; Mazina and Mazin, 2004; Petukhova et al.,
1998; Sigurdsson et al., 2002). It was found that this stimulation is dependent on
ATPase activity of Rad54 (Petukhova et al., 1998; Petukhova et al., 1999), suggesting that Rad54 translocation on dsDNA plays a role in stimulation of DNA strand exchange activity. It was proposed that the translocation activity of Rad54 may both provide a more efficient delivery of dsDNA to the site of the homology
24 search within the filament and the transient disruption of dsDNA base pairs as a consequence of DNA translocation may make them more available for interaction with the ssDNA bound within the Rad51 nucleoprotein filament. However, it was recently shown that ATP hydrolysis plays a minimal role in stimulation of Rad51 strand exchange activity (Deakyne et al., 2013). Therefore, further work is required to understand the exact mechanism for Rad54-depednent stimulation of Rad51’s
DNA strand exchange activity.
Furthermore, Rad54 was found to stabilize Rad51 nucleoprotein filament by increasing: i) the filament resistance against dissociation at elevated salt concentrations, ii) the Rad51-dependent protection of dsDNA against cleavages by restriction endonucleases, and iii) the Rad51 ability to compete with RPA for ssDNA binding (Wolner et al., 2003; Mazin et al., 2003). Nucleoprotein filament stabilization by Rad54 does not depend on its ATPase activity, as the Rad54
ATPase-deficient mutant is still proficient in Rad51-filament stabilization (Mazin et al., 2003). Consistent with this, S. cerevisiae strain expressing the ATPase- deficient rad54K341R, but not rad54∆ allele, was shown to be proficient in the recruitment of Rad51 to the site of DNA double-strand breaks (Wolner and
Peterson, 2005).
Paradoxically, Rad54 was also shown to disrupt the Rad51-dsDNA filament
(Figure 1.7) (Li et al., 2007; Solinger et al., 2002; Wright and Heyer, 2014). The active form of Rad51 is the nucleoprotein filament formed on ssDNA. However, by analogy with E. coli RecA protein (Pugh and Cox, 1987), it is likely that Rad51 remains associated with the dsDNA heteroduplex, the product of DNA strand
25 exchange. Therefore, Rad51 dissociation from duplex DNA would help (i) to recycle Rad51 for new rounds of recombination and prevent accumulation of cytotoxic nucleoprotein complexes (Shah et al., 2010), (ii) to free the nascent DNA joint in the D-loop from bound Rad51 permitting access of the primer end to a DNA polymerase to initiate repair DNA synthesis (Li and Heyer, 2009).
The Rad51-Rad54-ssDNA complex formed has significant and synergistic effects on the activities of both the proteins. While Rad54 stimulates the DNA strand exchange activity of Rad51 (Figure 1.7), in turn, Rad51 also stimulates the biochemical activities of RAD54, the dsDNA-dependent ATPase activity and DNA topology modification activity (generation of positive and negative supercoils)
(Mazin et al., 2000; Sigurdsson et al., 2002; Van Komen et al., 2000). Moreover,
Rad51 increases the processivity of Rad54 DNA translocation along DNA (Mazina and Mazin, 2004). Finally, Rad51 stimulates chromatin remodeling (Alexeev et al.,
2003; Alexiadis and Kadonaga, 2002; Jaskelioff et al., 2003; Kwon et al., 2007;
Zhang et al., 2007) and DNA branch migration activity of the Rad54 protein (Rossi and Mazin, 2008).
1.4.5 Rad54 Promotes Branch Migration of Holliday Junctions
Genetic data indicates the significance of post-synaptic (downstream of
Rad51) functions for Rad54 (Krogh and Symington, 2004; Rattray and Symington,
1994; Symington and Heyer, 2006). Evidence for the post-synaptic function of
Rad54 was provided by the analysis of rad54 srs2 double mutants. While the rad51
srs2 double mutant is viable, the rad54 srs2 double mutant is not (Palladino and
26
Klein, 1992). Furthermore, the synthetic lethality of the rad54 srs2 mutant is suppressed by rad51 mutation (Schild, 1995), indicating that Rad51-generated recombination intermediates are normally resolved by Rad54 and Srs2, but became lethal in the absence of these two proteins.
Figure 1.8 Rad54 promotes branch migration. Branch migration is the process in which one strand of the duplex DNA is progressively exchanged for another strand of the homologous duplex DNA and involves step-wise breakage and reformation of bonds. The cross-structure formed at the point of strand exchange is Holliday junction. In vitro, this structure is mimicked using oligonucleotide substrates that is able to branch migrate in presence of Rad54 protein.
Post-synaptic steps, after D-loop formation, include: 1) heteroduplex extension and formation of Holliday junctions, 2) priming of DNA synthesis from the invading 3’-OH end, 3) branch migration of Holliday junctions, 4) resolution of
Holliday junction intermediates and 5) sealing of the strands to restore two intact and contiguous duplex DNAs. There is evidence that Rad54 may play a role in several post-synaptic steps. It was shown that ScRad54 stimulates heteroduplex extension during DNA exchange promoted by ScRad51 in vitro (Solinger and
27
Heyer, 2001). Also, both ScRad54 and its meiosis-specific homologue ScRdh54 catalyze ScRad51 removal from dsDNA in an ATP-dependent fashion (Chi et al.,
2006; Solinger et al., 2002; Wright and Heyer, 2014), presumably to provide an access of DNA polymerases to the invading 3’-OH end for DNA synthesis (Li and
Heyer, 2009). Moreover, Rad54 binds efficiently to Holliday junctions and promotes their branch migration with high efficiency in an ATPase-dependent manner (Bugreev et al., 2006b; Mazina et al., 2007; Rossi and Mazin, 2008).
Finally, Rad54 stimulates the cleavage activity of Mus81-Eme1 (Mms4), a structure-specific endonuclease, which cleaves Holliday junction-like structures
(Matulova et al., 2009; Mazina and Mazin, 2008).
The formation of the cross structure during the process of homologous recombination (Figure 1.8), where the strands are exchanged between the two different DNA molecules was first predicted by Robin Holliday, and this structure is therefore known as Holliday junction (also called X-junction) (Holliday, 1964).
Holliday junction has the ability to branch migrate along the DNA axis by a process known as Branch migration, in which one DNA strand is progressively exchanged for another (Figure 1.8). Branch migration may extend or shorten the heteroduplex
DNA, formed after DNA strand invasion, thereby controlling the amount of genetic information transferred between the two DNA molecules (Pâques and Haber,
1999). It may cause dissociation of recombination intermediates and thereby affect the choice between a crossover and non-crossover pathway for recombination to proceed (Bugreev et al., 2007). There is genetic and biochemical data indicating the role of branch migration in re-starting stalled replication forks during cell
28 recovery after DNA damage (McGlynn et al., 2001; Michel et al., 2004; Postow et al., 2001; Rothstein et al., 2000; Seigneur et al., 1998).
Holliday junction structure has been analyzed by different methods including comparative gel electrophoresis, molecular modeling, fluorescence resonance energy transfer (FRET) assay, NMR and by X-ray studies (Lilley, 2000).
The Holliday junction may exist in the stacked or unstacked (extended) conformation. In the stacked conformation, pairs of helical arms stack coaxially to form nearly continuous criss-crossed duplexes, whereas, in the unstacked one, it forms a four-fold symmetric nearly planar structure. The presence of metal ions such as magnesium or calcium (at 100 µM and greater) neutralizes electrostatic repulsions caused by the phosphates, resulting in stacked conformation of Holliday junctions, whereas in the absence of metal ions, the DNA junction is fully extended.
The switch between the extended and stacked conformations is important for proteins acting on Holliday junctions. For example, RuvAB protein shows a binding preference to the Holliday junctions in the extended conformation (Ariyoshi et al.,
2000), whereas, hMSH4–hMSH5 complex, preferentially binds the Holliday junction in the stacked conformation (Snowden et al., 2004).
Branch migration occurs while the Holliday junction is in an extended state, whereas the stacked conformation is refractory for the branch migration process
(Karymov et al., 2005). A single step of branch migration requires the breakage of two base pairs at the point of strand exchange located on diametrically opposite arms, rotation around, and formation of the terminal base pairs of the remaining two arms. Single-molecule techniques were used to observe the rotation of DNA
29 strands during branch migration promoted by RuvAB (Han et al., 2006). The rate of spontaneous branch migration, when the Holliday junction is in the open conformation, is extremely fast with a step time of 300-400 µs (Panyutin and Hsieh,
1994), corresponding to a migration rate of 2500-3300 bp/sec, whereas, it is reduced to an estimated rate of about 3.3-3.7 bp/sec in the presence of magnesium (>100 µM) (Panyutin and Hsieh, 1994). The spontaneous branch migration proceeds bi-directionally making it inefficient under physiological conditions. Therefore, to provide directionality and processivity to the process of branch migration, there is a requirement for specialized enzymes.
In prokaryotes, RuvAB and RecG enzymes promote branch migration of
Holliday junctions (Liu and West, 2004; Lloyd and Buckman, 1991; Lloyd and
Sharples, 1993; Sharples et al., 1999; West, 1997). In eukaryotes, several branch migration proteins have been identified. Of all the known branch migration proteins,
RuvB belongs to the ATPases associated with diverse cellular activities (AAA+)
family (RuvB) and all the others belong to the SF2 helicase superfamily. These
SF2 proteins include: (1) RecG, a member of the RecG family; (2) several members of the RecQ helicase family, for example, BLM, WRN, RECQ1, and
RECQ5 in humans (Bugreev et al., 2008; Constantinou et al., 2000; Kanagaraj et
al., 2006; Karow et al., 2000; Wu et al., 2006); (3) FANCM, a member of the DEAH
helicase family; and (4) Rad54, a member of the Snf2 family of DNA translocases.
Despite structural diversity among branch migration proteins belonging to
different families, all of these proteins i) are capable of translocation on DNA in an
ATPase-dependent manner, and ii) show high binding affinity to the Holliday
30 junction (or other cruciform DNA structures that are capable of branch migrating).
These two functions can either be segregated, for example in RuvAB complex, where RuvA protein is structure-specific DNA binding protein and RuvB protein is a motor protein (West, 1997). Or these functions can be combined in a single polypeptide, like in RecQ helicase family members, BLM, WRN, or RecQ1, that have a motor domain and a separate structure-specific DNA-binding region located in their C-terminus (Hickson, 2003). In the Rad54 protein, the domain(s) responsible for binding to the Holliday junction is yet to be identified.
Similar to other branch migration proteins, Rad54 translocates on dsDNA in an ATPase-dependent manner (Amitani et al., 2006) and binds efficiently to synthetic Holliday junctions (Bugreev et al., 2006b; Mazina et al., 2007). Rad54 shows higher affinity for the X-junction and the partial X-junction (PX-junction), approximately 30-fold and 200-fold respectively, compared to dsDNA. The PX- junction is an appropriate substrate for DNA branch migration proteins as it resembles one end of a D-loop, the product of DNA strand invasion (Figure 1.8).
The branch migration activity of Rad54 was demonstrated using a fully movable
X-junction and was found to be ATPase-dependent (Bugreev et al., 2006a;
Bugreev et al., 2006b). In addition to 4-stranded branch migration, Rad54 catalyzes 3-stranded branch migration of PX-junction and of plasmid-size DNA substrates. It was found that Rad54 protein efficiently promotes branch migration
Holliday junctions through DNA regions of several thousand base pairs (Figure
1.8) (Bugreev et al., 2006b) and that the rate of branch migration is comparable with that of RuvAB, a classical branch migration protein from E.coli.
31
The “minimal” branched DNA substrate that efficiently stimulates ATP hydrolysis consists of three DNA arms, two short dsDNA arms, 15 bp each, and a ssDNA arm of 45 nt (Mazina et al., 2007), which is consistent with the crystallographic data showing that the S. solfataricus Rad54 catalytic core domain can accommodate a 15 bp dsDNA fragment (Durr et al., 2005). HRad54 titrations using the “minimal” DNA substrate showed that the stoichiometry for Rad54's
ATPase activity is two protein monomers per DNA molecule (Mazina et al., 2007).
However, the branch migration was relatively low at dimer concentrations and reached a maximum at a concentration of 10 ± 2 monomers per DNA molecule indicating the requirement for higher oligomeric structures for branch migration.
The difference in the stoichiometry for ATPase and branch migration activities suggests that either the additional Rad54 monomers have an ATPase- independent DNA binding activity or are involved in Rad54 protein-protein interactions essential for branch migration activity. Previously, it was shown that different multimeric forms of RECQ1 are associated with different enzymatic activities of the protein (Muzzolini et al., 2007).
1.5 ROLE OF RAD54 BRANCH MIGRATION ACTIVITY IN HOMOLOGOUS
RECOMBINATION
During the initial steps of homologous recombination, Rad51-promoted joint
molecule (D-loop) formation takes place. Then, the 3’ end of the invading ssDNA
is extended by DNA polymerase, retrieving the information lost at the site of the
break. Following this, the homologous recombination may proceed through either
32 of the two pathways (Allers and Lichten, 2001; Hunter and Kleckner, 2001). The extended joint molecules could either dissociate, leading to rejoining of the broken chromosome through synthesis-dependent strand annealing (SDSA) (Allers and
Lichten, 2001), or they proceed through capture of the second processed DNA end, producing single or double Holliday junctions (Cromie et al., 2006; Schwacha and Kleckner, 1995), which can later be resolved by structure-specific endonucleases (Pâques and Haber, 1999). The joint molecules (D-loops) dissociation after the completion of template-dependent DNA synthesis is an essential step of double-strand break repair via the SDSA mechanism (Pâques and Haber, 1999). It has been shown that Rad54 promotes dissociation of D-loops formed by Rad51 protein owing to its ATP-dependent branch-migration activity.
More importantly, Rad54 can also dissociate D-loops containing Rad51 protein, which mimics in vivo recombination intermediates.
Consistent with this, there is genetic data that Rad54’s D-loop dissociation activity contributes to its function during the late stages of homologous recombination. Specifically, the D-loops dissociation ability of Rad54 in vitro agrees with the reduced length of gene conversion tracts in yeast strains overexpressing Rad54 protein (Kim et al., 2002).
Rad54 can also dissociate double D-loops (Bugreev et al., 2007), generated by Rad52 through the annealing of the second end of broken DNA to the single D- loops (Figure 1.1) (Bugreev et al., 2007; McIlwraith and West, 2008; Nimonkar et al., 2009; Sugiyama et al., 2006). The double D-loops dissociation by Rad54 occurs through a mechanism, in which D-loop dissociation is directly associated to
33 rejoining of the broken DNA ends without a reannealing step (Bugreev et al., 2007).
Undissociated double D-loops can lead to the formation of double Holliday junctions and then crossovers, which might lead to detrimental loss of heterozygosity and lead to genome instability in somatic cells. Therefore, double
D-loop dissociation by Rad54 may play an important role by promoting homologous recombination via non-crossover pathways. Thus, DNA branch migration activity of the Rad54 protein may play a part in the double-stranded break repair mechanism postulated by the SDSA model (Pâques and Haber,
1999).
1.6 RESOLUTION OF HOLLIDAY JUNCTION
The interaction between Rad54 protein and Mus81, a structure-specific
endonuclease, is evolutionarily conserved in eukaryotes (Hanada et al., 2006;
Interthal and Heyer, 2000; Matulova et al., 2009; Mazina and Mazin, 2008; Mimida et al., 2007). Mus81 belongs to XPF/MUS81 family of nucleases that share a highly
conserved motif (V/IERKX3D) constituting an integral part of the endonuclease
catalytic site. Mus81 functions as a heterodimer with a non-catalytic partner protein
known as Eme1 in fission yeast and humans or as Mms4 in budding yeast and
Drosophila (Ciccia et al., 2008). The role of Mus81 in the resolution of Holliday
junctions was first proposed in studies on S. pombe, where all the defects of mus81
mutants could be rescued by expression of RusA, a bacteriophage resolvase that
is highly specific for Holliday junctions (Blais et al., 2004; Boddy et al., 2001; Boddy
et al., 2000). The Holliday junction is a substrate for Mus81, responsible for its
34 cleavage and formation of crossover products to complete the double-strand break repair process (Heyer, 2004; Liu and West, 2004).
Rad54 interacts with Mus81-Eme1 (Mms4) and stimulates the DNA cleavage activity of Mus81-Eme1 (Figure 1.9) (Matulova et al., 2009; Mazina and
Mazin, 2008). This stimulation shows a functional link between Rad54, a branch migration protein, and Mus81–Eme1 (Mms4), a structure-specific endonuclease.
The observations that Mus81-Eme1 stimulation requires the addition of Rad54 to
DNA-junctions first or, at least concurrently with Mus81-Eme1, suggest that Rad54 targets Mus81 to DNA junctions via protein-protein interactions (Matulova et al.,
2009; Mazina and Mazin, 2008).
Figure 1.9 Rad54 stimulates endonuclease activity of Mus81. The DNA intermediates formed during homologous recombination need to be resolved to complete the repair process. Rad54 interacts with Mus81 and stimulates endonuclease activity of Mus81-Eme1, a structure-specific endonuclease that recognizes branched DNA substrates.
35
Stimulation of Mus81-Eme1 activity by Rad54 in vitro is consistent with genetic data in S. cerevisiae (Interthal and Heyer, 2000; Matulova et al., 2009) and in mouse ES cells (Hanada et al., 2006). Taken together, these results suggest that Rad54 and Mus-81 may cooperate in the processing of Holliday junctions intermediates formed during the repair of double-strand breaks or stalled replication forks.
1.7 REGULATION OF RAD54 ACTIVITIES
Homologous recombination pathway helps in maintaining genome stability.
However, if it is not controlled or regulated, it could lead to loss-of-heterozygosity,
genomic rearrangements, and toxic recombination intermediates leading to cell
death (Heyer et al., 2010). Therefore, regulation of homologous recombination
needs to be carefully monitored in the cell. A good target for the regulation of
homologous recombination pathway is Rad54, which is a versatile protein functioning throughout the process of homologous recombination. In S. pombe,
Rhp54 is targeted for ubiquitin-mediated proteolysis to (1) down-regulate homologous recombination in the G1 phase, (2) shift the recombination bias from inter-sister to inter-homolog during meiosis (Trickey et al., 2008).
On the other hand, in S. cerevisiae, ScRad54 is regulated by Mek1 (a meiosis-specific kinase) phosphorylation at T132, shown both in vitro and in vivo
(Niu et al., 2007; Niu et al., 2009). The phosphorylation of ScRad54 at T132 is required to bias the meiotic recombination towards inter-homolog instead of inter- sister interactions (Niu et al., 2007). The T132D phosphomimetic mutation in
36
ScRad54 showed significantly weak interaction with ScRad51, thereby reducing the stimulation of ScRad54’s ATPase activity by ScRad51 as well the D-loop formation by ScRad51 when compared to wild-type ScRad54 (Niu et al., 2009).
The interaction between ScRad54 and ScRad51 is also regulated during meiosis by Hed1, a meiosis-specific Rad51 binding protein that prevents binding of
ScRad54 to ScRad51 (Busygina et al., 2008; Tsubouchi and Roeder, 2006). Hed1 also inhibits the interaction of ScRad51 with ScRdh54, though to a lesser extent compared to ScRad54 (Busygina et al., 2008). The inhibition of ScRad54 functions during meiosis is believed to be transient as ScRad54 is required for successful meiosis.
Recently, it was shown that cell cycle phase-specific phosphorylation of hRad54 by Nek1 at Ser-572 promotes removal of hRad51 in the late G2 phase prior to the onset of mitosis and completion of homologous recombination (Spies et al., 2016). Several post-translational modifications have also been identified in human Rad54 (Kettenbach et al., 2011; Li et al., 2009; Mertins et al., 2014; Sharma et al., 2014), with most of these modifications occurring in the N-terminal domain of Rad54. The N-terminal domain of Rad54 is not well conserved amongst orthologs, probably to enable species specific interactions with partner proteins.
Therefore, post translational modifications occurring in the Rad54 N-terminal domain could regulate its interaction with different proteins during the process of homologous recombination. Clearly, further work is needed to shed light on how the different functions of Rad54 are tightly regulated in the cell.
37
1.8 PROTEIN REGULATION BASED ON ITS OLIGOMERIC STATE
Protein oligomerization is common and takes place in all biological systems.
An estimated 35% of all the proteins in a cell are oligomeric (Goodsell and Olson,
2000). The oligomeric proteins could either be homo-oligomers or hetero-
oligomers, ranging from dimers to higher order oligomers (Gabizon and Friedler,
2014). The different oligomeric states of the protein could be associated with
different activities, and switching between these states may help the protein
regulate its functions. For example, RecQ1 helicase exhibits DNA unwinding in the
monomer or tightly bound dimer form, whereas, the higher-order oligomers of
RecQ1 have DNA strand annealing activity (Muzzolini et al., 2007). Therefore,
correct protein oligomerization is critical for its function and is tightly regulated by
various factors. Protein oligomerization could be regulated by the binding of
partner proteins, nucleotide cofactors, metal cofactors, or by binding to DNA etc.
In the example of RecQ1 helicase, the presence of ssDNA favors formation of
higher order oligomers whereas, the presence of ATP or ATPγS shifted the
equilibrium towards smaller oligomeric species (Muzzolini et al., 2007).
Rad54 protein exists as a monomer in solution but rapidly undergoes
oligomerization in presence of DNA (Petukhova et al., 1999). The stoichiometry for
Rad54's ATPase activity was shown to be two protein monomers per DNA
molecule (Mazina et al., 2007), whereas, the branch migration reached a maximum
at a concentration of 10 ± 2 monomers per DNA molecule indicating the
requirement for higher oligomeric structures for branch migration (Mazina et al.,
2007). Thus, like other proteins, Rad54 has functions that are dependent on its
38 oligomeric state. Therefore, it would be interesting to determine 1) the Rad54 domain that regulates its DNA-dependent oligomeric state and 2) the role oligomerization plays in modulating Rad54’s activities.
Because oligomerization is common, and crucial for the activity of many proteins, there is an increasing interest to develop small-molecule compounds that would modulate the state of protein oligomerization. The protein oligomerization could be modulated by binding of the small-molecules directly to the oligomerization interface or by stabilizing the protein in a specific oligomeric state.
A number of compounds already in clinical use were later discovered to act by modulating protein oligomerization. For example, the anti-cancer drug, Taxol, binds to β-tubulin and allosterically inhibits assembly and disassembly dynamics of microtubules (Derry et al., 1995; Wani et al., 1971). Therefore, modulating protein oligomerization provides a promising target for therapeutic intervention.
1.9 SMALL-MOLECULE INHIBITORS OF HOMOLOGOUS RECOMBINATION
PROTEINS
Tumorigenesis is the uncontrolled growth of cells that are able to avoid cell
death mechanisms (Bartkova et al., 2005). This uncontrolled and often rapid
proliferation of cells can lead to benign tumors, and some of these may develop
into malignant tumors (cancers). In the past, cancer therapy has relied on the use
of a combination of radiation with toxic chemotherapeutic agents for several years.
This approach, though successful in experimental settings, has not been so
successful from the clinical point of view (Russell et al., 2003). The rapid and
39 uncontrolled growth of cancer cells increases the stress on DNA damage and replication pathways, making them “addicted” to DNA-damage repair pathways for survival (Curtin, 2012). It is believed that the DNA repair proteins are regulated differently in cancer cells compared to normal cells. In many cancers, the expression of DNA repair proteins is upregulated or dysfunctional. In many tumor cell lines, the Rad51 mRNA and Rad51 protein levels were increased by 11-fold and 7-fold, respectively, compared to normal cell lines (Raderschall et al., 2002).
On the other hand, some studies found a decrease in Rad51 protein levels in tumors (Takaku et al., 2011) suggesting deregulation of the Rad51 levels in tumors. Additionally, the expression of Rad54, and Rad51 associated protein 1
(RAD51AP1) was on an average 4-fold and 2-fold higher, respectively, in BRCA1 deficient tumors (Martin et al., 2007). Taken together, these results indicate upregulation of homologous recombination pathway in tumors as means of surviving the DNA damage caused by either rapid cell growth or by toxic chemotherapeutic agents used for cancer therapy. Therefore, new cancer therapies targeting homologous recombination proteins, in addition to the use of chemotherapeutic agents that cause DNA damage is the need of the hour.
The use of small molecule compounds to modulate the activity of proteins has increased over the past few years because of the ease with which they could be applied and removed over a controlled time period. Another advantage of using small molecules besides being used as drugs to treat specific diseased conditions is to understand the activities or functions of a protein in the cell. For example, B02 inhibits Rad51 recombinase and has been shown to potentiate the effect of
40 cisplatin used for the treatment of cancer. Also, streptonigrin, an inhibitor of Rad54
ATPase activity, was used to study the mechanistic differences between two different activities of Rad54: stimulation of strand exchange and branch migration.
In addition to these, several other small-molecule inhibitors targeting different homologous recombination proteins have been identified.
Given the malleable and heterogeneous nature of cancer cells, one drawback of using small-molecule compounds is that prolonged use over time could result in the development of resistant cancer cells. Cancer cells can develop resistance via various mechanisms like by mutating or amplifying the drug target, genetic alterations to avoid cell death wherein, or by acquiring features enabling them to survive under selective drug pressure. In addition, cells may also become resistant either by altering the drug transport or metabolism.
Hence, there is a need for new therapies and patient-specific therapies to successfully eradicate cancer cells. One strategy could involve inhibition of specific
DNA repair proteins and knowing the DNA damage response status of a particular tumor in order to induce synthetic lethality in cancer cells. For this, continuing research is necessary to fully understand the homologous recombination process in normal cells versus tumor cells and how the multiple DNA repair pathways are coordinated to carry out the repair.
41
1.10 SUMMARY
Homologous recombination plays a critical role in repair of the most harmful
types of DNA lesions, DNA double-strand breaks, and interstrand cross-links, in
faithful segregation of homologous chromosomes during meiosis, and in telomere maintenance. Failure to repair double-strand breaks due to inefficient homologous recombination results in the accumulation of chromosomal aberrations as well as mutations, leading to genome instability, and predisposition to cancers, and other disorders like Bloom’s Syndrome, Werner’s syndrome, and Fanconi Anemia.
Rad54 is one of the key proteins of homologous recombination and is evolutionarily conserved. It is a motor protein that translocates on duplex DNA in an ATPase-dependent manner. Rad54 is a multifunctional protein that by interacting with different protein partners throughout the process of homologous recombination, helps in linking together early and late steps of homologous recombination. The transition from the early function of Rad54 in stimulating Rad51 to branch migration of Holliday junctions during late stages of homologous recombination remains to be understood. In other words, how the different activities of Rad54 during homologous recombination are regulated, is not yet known.
Therefore, for my Ph.D. dissertation, we focused on understanding what regulates Rad54’s activities and the mechanism for branch migration of Holliday junctions. We wanted to determine the domain in Rad54 that is responsible for
DNA-dependent oligomerization and the role of oligomerization in regulating
42
Rad54 activities. Here, we demonstrate that the N-terminal domain of Rad54 is essential for its DNA branch migration activity along with the conserved ATPase or “core” domain.
Using N-terminal truncated Rad54 and different mutants of Rad54, we show that a second DNA-binding site in the N-terminal domain is essential for DNA branch migration activity of Rad54. This second DNA-binding site is dispensable for other Rad54 activities such as ATP hydrolysis and translocation. We found that this second DNA-binding site has a preference for branched DNA structures and
DNA binding by this site enables oligomerization of Rad54. We also identified a specific amino acid Ser-49, important for oligomerization. Also, CDK2 dependent phosphorylation of Rad54 at Ser-49 in vitro or the phosphomimetic mutation,
S49E, impairs the ability of Rad54 to oligomerize and inhibits its branch migration activity without affecting its ATP hydrolysis or the ability to stimulate strand exchange activity of Rad51. Taken together, our results show for the first time that the N-terminal domain plays a pivotal role in the assembly of the active Rad54 branch migration complex. Also, CDK2-dependent phosphorylation of hRad54 at
Ser-49 may help in regulating two important functions of Rad54: stimulation of
Rad51’s strand exchange activity and branch migration of Holliday junctions.
For future studies, we would be interested in determining the specific role of branch migration activity of Rad54 in the process of homologous recombination by expressing hRad54S49E and hRad54S49A mutants in cells. Furthermore, we show
that by targeting N-terminal domain of Rad54, it is possible to specifically inhibit
one of its functions without affecting the others. This could have a potential
43 application in drug development, as inhibitors of homologous recombination proteins have shown to potentiate the effects of anti-cancer drugs.
44
RAD54 N-terminal domain: a DNA sensor that couples ATP hydrolysis with branch migration of Holliday junctions
2.1 ABSTRACT
In eukaryotes, RAD54 protein plays an important role in homologous
recombination. RAD54 is a Swi2/Snf2 ATP-dependent DNA translocase that
promotes branch migration of Holliday Junctions, chromatin remodeling, and
stimulates DNA strand exchange activity of RAD51. The branch migration activity
of RAD54 is the strongest among eukaryotic proteins. However, despite extensive
studies, the mechanism of this activity is not well understood. Here, we
demonstrate that the RAD54 N-terminal domain is essential for branch migration
along with the conserved ATPase/DNA binding domain. Our results demonstrate
that the N-terminal domain is responsible for initiation of branch migration through
two connected, but distinct steps; the N-terminal domain binds to Holliday
Junctions with high affinity, and the binding event triggers RAD54 multimerization
that is essential for branch migration. We identified the RAD54 N-terminal domain mutations that separately affect DNA binding and multimerization by N-terminal domain. We also show that the RAD54 N-terminal domain may play a regulatory
role serving as a target for CDK2; S49 phosphorylation by CDK2 down regulates
branch migration activity by attenuating DNA-dependent multimerization of
RAD54.
45
2.2 INTRODUCTION
The homologous recombination pathway is responsible for the repair of
DNA double-strand breaks, the most harmful types of DNA lesions, faithful
chromosome segregation during meiosis, and telomerase-independent telomere maintenance (Jasin and Rothstein, 2013; Krejci et al., 2012; San Filippo et al.,
2008). Homologous recombination uses homologous DNA molecules as a template to repair double-strand breaks and therefore is, generally, an error-free process. During double-strand break repair by homologous recombination, the dsDNA ends undergo exonucleolytic resection to generate protruding ssDNA tails
(Symington, 2014). RAD51 recombinase binds to these ssDNA tails forming a nucleoprotein filament that performs a search for homologous dsDNA
(Kowalczykowski, 2008). The RAD51-ssDNA filament then invades the homologous dsDNA generating joint molecules also known as D-loops. D-loops further extend into the DNA four-way cross-structure that is known as a Holliday
Junction (Holliday, 1964; Mazin et al., 2010; Wyatt and West, 2014). The Holliday
junction has a remarkable ability to translocate along the DNA axis through a
process known as branch migration, in which one strand of the DNA duplex is
progressively exchanged for the homologous strand of another DNA duplex by the
stepwise breakage and reformation of base pairs. Branch migration may cause
either dissociation or extension of joint molecules, depending on the specific
homologous recombination mechanism. It may also promote a restart of DNA
replication stalled at a DNA damage site by switching DNA template strands
through a reversible regression of replication forks into Holliday junctions.
46
Previously, we showed that RAD54, a member of the Rad52 epistasis group
(Game, 2000; Krogh and Symington, 2004), promotes branch migration of Holliday junctions. RAD54 is a multifunctional protein that, in addition to branch migration, can physically interact with RAD51 (Golub et al., 1998; Raschle et al., 2004) to stimulate its DNA strand exchange activity (Mazina and Mazin, 2004; Petukhova et al., 1998; Sigurdsson et al., 2002). RAD54 was also shown to remodel nucleosomes (Alexeev et al., 2003) and displace RAD51-dsDNA complexes
(Solinger et al., 2002).
RAD54 promotes branch migration with significantly greater efficiency than other known eukaryotic branch migration proteins, like BLM or RECQ1 (Bugreev et al., 2006b; Mazina et al., 2012). Moreover, similar to RuvAB, a canonical branch migration protein from E.coli, RAD54 is capable of driving branch migration of
Holliday junctions, through large regions of DNA sequence heterology (Mazin et al., 2010; Mazina et al., 2012). Previous studies showed that the branch migration activity of RAD54 requires ATP hydrolysis and involves the formation of higher order RAD54 oligomers on Holliday junction-like structures (Bugreev et al., 2006b;
Mazina et al., 2007). However, the mechanism of RAD54 branch migration is currently not well understood.
RAD54 belongs to the SWI/SNF2 family of helicase-like proteins of
Superfamily (SF) 2 helicases (Gorbalenya and Koonin, 1993; Laurent et al., 1993).
Similar to other SWI/SNF proteins, but unlike canonical helicases, RAD54 does not have DNA strand separation activity. Structurally, RAD54 is composed of the
N-terminal domain, the ATPase/DNA binding core domain, and the C-terminal
47 domain. The ATPase/DNA binding core domain contains seven motifs conserved in all SF2 and SF1 proteins, I, Ia, II, III, IV, V and VI that constitute the two RecA- like domains responsible for protein binding to DNA and translocation in an
ATPase-dependent manner (Caruthers and McKay, 2002; Singleton et al., 2007;
Thoma et al., 2005). The RAD54 N-terminal domain and C-terminal domain are unique for RAD54 orthologues and their functional role remains to be elucidated.
In this study, using the N-terminal truncated RAD5496-747, we found that the N-
terminal domain of RAD54 is essential for its branch migration activity. At the same
time, the RAD5496-747 retains the ability to translocate on dsDNA in an ATPase-
dependent manner, indicating a specific role of the RAD54 N-terminal domain in
branch migration. We found that a distinct DNA binding site located in the N-
terminal domain (Wright and Heyer, 2014) is critical for RAD54 branch migration.
First, this site shows a strong preference for Holliday junction and Holliday junction-
like structures. Second, DNA binding by this site enables protein multimerization
that is required for branch migration. We identified a specific amino acid residue,
S49, which is important for protein multimerization. Furthermore, we show that
CDK2 phosphorylation at S49 in vitro or the S49E phosphomimetic mutation
impairs DNA-dependent RAD54 oligomerization and inhibits RAD54 branch
migration activity. Taken together, these data indicate that the N-terminal domain
may play a pivotal role in assembly of an active RAD54 branch migration complex
and in the regulation of RAD54 branch migration activity.
48
2.3 Materials and Methods
Proteins and DNA
All oligonucleotides used in this study (Table 2.1) were purchased from IDT
Inc and purified using denaturing 6-10% PAGE. To prepare oligonucleotide dsDNA
substrates, complementary ssDNA oligonucleotides were annealed as described
(Rossi et al., 2010) and stored at -20 °C. Oligonucleotides were labeled using [γ-
32P] ATP and T4 polynucleotide kinase. T4 polynucleotide kinase and restriction endonucleases were purchased from New England Biolabs. PreScission™
Protease was purchased from GE Healthcare Life Sciences. CDK2/Cyclin E complex was a generous gift of Bruce Clurman (Fred Hutchinson Cancer Research
Center). The CDK2/CyclinA2 complex was purchased from ProQinase GmbH
(Germany).
Table 2.1 Sequences of the oligonucleotides used in this study *
Length in Number Sequence, 5’3’ nucleotides
GTC GAC GAC GTC TGA GTA CTC ATC TAG TGT GAC ATC #64 48 ATC GCA TCG AGA
TCT CGA TGC GAT GAT GTC ACA CTA GAT GAG TAC TCA #65 48 GAC GTC GTC GAC
CTT TAG CTG CAT ATT TAC AAC ATG TTG ACC TAC AGC #71 94 ACC AGA TTC AGC AAT TAA GCT CTA AGC CAT CCG CAA AAA TGA CCT CTT ATC AAA AGG A
TCC TTT TGA TAA GAG GTC ATT TTT GCG GAT GGC TTA #169 93 GAG CTT AAT TGC TGA ATC TGG TGC TGT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT
49
T CCT TTT GAT AAG AGG TCA TTT TTG CGG ATG GCT TAG #170 94 AGC TTA ATT GCT AAA TCT GGT GCT GTA GGT CAA CAT GTT GTA AAT ATG CAG CTA AAG
ACA GCA CCA GAT TTA GCA ATT AAG CTC TAA GCC ATC #171 63 CGC AAA AAT GAC CTC TTA TCA AAA GGA
#174 61 GAC GCT GCC GAA TTC TAC CAG TGC CTT GCT AGG ACA TCT TTG CCC ACC TG CAG GTT CAC CC
#175 62 TGG GTG AAC CTG CAG GTG GGC AAA GAT GTC CTA GCA ATG TAA TCG TCA AGC TTT ATG CCG TT
#176 63 CAA CGG CAT AAA GCT TGA CGA TTA CAT TGC TAG GAC ATG CTG TCT AGA GGA TCC GAC TAT CGA
#177 62 ATC GAT AGT CGG ATC CTC TAG ACA GCA TGT CCT AGC AAG GCA CTG GTA GAA TTC GGC AGC GT
#180 25 CTT TGC CCA CCT GCA GGT TCA CCC A
#181 25 TCG ATA GTC GGA TCC TCT AGA CAG C
A CAT TGC TAG GAC ATG CTG TCT AGA GGA TCC GAC TAT #244 60 CGA TAA AAC CCT GCA AGT TCG TA
#249 30 TG CAG GTG GGC AAA GAT GTC CTA GCA ATG T
#250 15 CTT TGC CCA CCT GCA
TFO 22 TTC TTT TCT TTC TTC TTT CTT T
*Bold red letters indicate the nucleotides that form mismatched pairs in the branch migration products.
Purification of RAD54 and N-terminal truncated mutants from insect cells
Human RAD54 and RAD5496-747 were purified from Sf21 cells as described previously (Mazina and Mazin, 2004) with alterations indicated below. We used
RAD54 constructs with an N-terminal GST tag and a PreScission Protease™
50 recognition site (LEVLFQGP). Following fractionation on a Glutathione Sepharose and a Superdex 200 column, the RAD54 pool was incubated with PreScission
Protease (GE Healthcare) (10 U/100 mg of RAD54) for 4 h at 4 °C. The sample was diluted to 100 mM KCl and loaded on a 1 ml Resource S column. The fractions containing RAD54 or RAD54 mutants were analyzed for nuclease contamination, pooled, and stored in small aliquots at -80 °C. The protein appeared nearly homogeneous in a Coomassie-stained SDS-polyacrylamide gel.
Expression and purification of RAD54, and RAD54 mutant proteins from
E.coli
T31E S49E T31E Human RAD54, RAD54 , RAD54 , RAD541-142, RAD541-142 ,
S49E RKRK33-36AAAA RK52-53AA RKRK33-36AAAA RAD54 1-142 , RAD541-142 , RAD541-142 , RAD54 ,
and RAD54RKRK33-36AAAA, S49E with a Sumo-His affinity tag, were cloned in the
pETHSUL vector (Weeks et al., 2007) and expressed in E. coli Rosetta 2 cells.
The cDNA sequence used for cloning was amplified using DNA primers listed in
Table 2.2 and pFastBac-HTc vector containing RAD54 cDNA as template. The
cloning was performed using Ligation independent cloning protocol as described
previously (Weeks et al., 2007). All RAD54 mutants were generated using
QuickChange site directed mutagenesis kit (Agilent Technologies) and DNA
primers listed in Supplemental Table 2. Rosetta 2 cells were transformed with the
recombinant plasmids and cultured at 37 °C to an O.D.600 of 0.4. The cultures were transferred to 16 °C and allowed to grow until an O.D.600 of 0.6-0.8, followed by
induction of protein expression with 0.1 mM isopropyl-1-thio-β-D-
51 galactopyranoside for 20 h. Cells were harvested by centrifugation (7000 x g) and stored at -80oC. All purification steps were carried out at 4 °C. Cells (10 g) were
thawed and resuspended in ten volumes of lysis buffer (20 mM KH2PO4 pH 7.5,
200 mM KCl, 10% Sucrose, 10 mM 2-mercaptoethanol) supplemented with EDTA- free protease inhibitor cocktail (Roche Applied Science). The cells were lysed by passing their suspension twice through an Emulsiflex C-5 (Avestin) at 15, 000 –
20, 000 psi. The crude extract was clarified by centrifugation (148, 000 X g for 60 min) and passed through 0.45 µm filter. The filtrate was loaded onto a 5 ml HiTrap
Ni2+ column (GE Healthcare). The column was washed extensively with buffer A
(20 mM KH2PO4 pH 7.5, 500 mM KCl, 10% glycerol, 10 mM 2-mercaptoethanol)
supplemented with 50 mM Imidazole. RAD54 protein was eluted using buffer A
supplemented with 500 mM Imidazole and the eluate was supplemented with 2
mM EDTA. Sumo-His tags were cleaved off by Sumo hydrolase (dtUD1) (200 µg)
for 4 h at 4 °C. The cleaved RAD54 was fractionated on a Superdex 200 column
(58 ml) equilibrated with buffer A. The fractions containing RAD54 were pooled,
diluted 5 times using buffer B (20 mM KH2PO4 pH 7.5, 10% glycerol, 10 mM 2-
mercaptoethanol) and loaded on to 1 ml Resource S column (GE Healthcare), equilibrated with buffer B supplemented with 100 mM KCl. The proteins were eluted with a 20 ml gradient of KCl (100-450 mM) in buffer B. The fractions containing RAD54 or RAD54 mutants were analyzed for nuclease contamination, pooled, and stored in small aliquots at -80 °C. The protein appeared nearly
homogeneous in a Coomassie-stained SDS-polyacrylamide gel.
52
The RAD541-142 was purified as above except that a 58-ml Sephacryl S-200
HR column (GE Healthcare) was used in place of Superdex 200. For the RAD541-
142 mutants, 1 ml Heparin column was used instead of Resource S column.
Table 2.2 Primers used in this study *
Forward/R Sequence, 5’3’ everse
AG ATT GGT GGC GGC ATG AGG AGG AGC TTG GCT CCC Forward RAD54 WT
GAG GAG AGT TTA GAC ATT AGC GGA GGC CCC GCT GTT Reverse CCT CAT G
Forward AG ATT GGT GGC GGC ATG AGG AGG AGC TTG GCT CCC
RAD541-142 Reverse GAG GAG AGT TTA GAC A TTA A TGG ACA GGG AGT TTC TCC TTG
Forward G GAG TGT TTC CTG GAA CCT TTT CGG AAA CCT TTG AG RAD54 S49E Reverse CT CAA AGG TTT CCG AAA AGG TTC CAG GAA ACA CTC C
Forward CCC AGC CAG CTG GCC GCG GCA GCA CCT GAA GGC AGG TCC RAD54KRK12- 14AAA Reverse GGA CCT GCC TTC AGG TGC TGC CGC GGC CAG CTG GCT GGG
CCT GGC CTA GTG ACT CCT GCG GCA GCG GCA TCC Forward AGC AGT GAG ACC C RAD54RKRK33- 36AAAA Reverse GGG TCT CAC TGC TGG ATG CCG CTG CCG CAG GAG TCA CTA GGC CAG G
53
GAG TGT TTC CTG TCT CCT TTT GCG GCA CCT TTG AGT Forward CAG CTA ACC RAD54RK52-53AA Reverse GGT TAG CTG ACT CAA AGG TGC CGC AAA AGG AGA CAG GAA ACA CTC
*Bold red letters indicate the nucleotides that were mutated.
Spectrophotometric ATPase assay
The hydrolysis of ATP by Rad54 protein was monitored
spectrophotometrically as described (Kowalczykowski and Krupp, 1987). The
oxidation of NADH, coupled to ADP phosphorylation, resulted in a decrease in
absorbance at 340 nm, which was continuously monitored by a Hewlett-Packard
8453 diode array spectrophotometer using UVvisible ChemStation software. The
rate of ATP hydrolysis was calculated from the rate of change in absorbance using
- the following formula: the rate of A340 decrease (s 1) X 9880 = rate of ATP
hydrolysis (µM/min). The reactions were carried out in standard buffer containing
25 mM Tris-acetate, pH 7.5, 10 mM MgCl2, 1 mM DTT, 2 mM ATP, 3 mM
phosphoenolpyruvate, pyruvate kinase (20 U/ml), lactate dehydrogenase (20
U/ml), and NADH (200 μg/ml) unless otherwise indicated, and the indicated
concentrations of RAD54 and DNA. Reactions were carried out at 30 °C unless
otherwise indicated.
54
Branch migration assay
PX junctions were prepared by annealing 32P-labeled forked DNA
intermediates (#71/169*) with 3’-tailed DNAs (#170/171) (50% molar excess of
cold tailed DNA) (Rossi et al., 2010). The RAD54 protein (60 nM, unless indicated
otherwise) was incubated with 32P-labeled synthetic PX-junction (32 nM,
molecules unless indicated otherwise), in a 100-µl branch migration buffer
containing 25 mM Tris acetate, pH 7.5, 3 mM magnesium acetate, 2 mM ATP, 1
mM dithiothreitol, 100 µg/ml BSA, the ATP-regenerating system (30 units/ml
creatine phosphokinase and 10 mM phosphocreatine). The reactions were carried
out at 30 °C. Aliquots (10 µl) were withdrawn at indicated time points, and DNA
substrates were deproteinized by treatment with stop solution (1.36% SDS, 1.4 mg/ml proteinase K, 6% glycerol, 0.015% bromophenol blue) for 15 min at 37 °C.
Samples were analyzed by electrophoresis in 8% polyacrylamide gels (29:1) in 1x
TBE buffer (90 mM Tris borate, pH 8.3, and 1 mM EDTA) at room temperature.
The gels were dried on DE81 chromatography paper (Whatman) and quantified
using a Storm 840 PhosphorImager (GE Healthcare).
Triple-helix displacement assay
The assay was a modification of a procedure described previously (Firman
and Szczelkun, 2000; Jaskelioff et al., 2003). SspI-linearized pMJ5 (100 nM, molecules) and 32P-labeled triplex-forming oligonucleotide (TFO) (oligo TFO, 22- mer) (100 nM, molecules) were mixed in buffer containing 25 mM MES (pH 5.5), and 10 mM MgCl2 and incubated for 15 min at 57 °C, followed by overnight
55 incubation at room temperature. The RAD54 (5 nM) was incubated for 5 min at 20
°C in 100-µl reaction buffer containing 35 mM Tris HCl, pH 7.2, 3 mM MgCl2, 100
µg/ml BSA, 1 mM DTT, 2 mM ATP, and the ATP-regenerating system (30 units/ml creatine phosphokinase and 15 mM phosphocreatine). The reaction was initiated by addition of the triple-helix substrate (pMJ5+TFO) (0.5 nM, molecules). 10 µl aliquots were withdrawn at indicated time points and the reaction was quenched by adding 5 µl of stop solution containing 1.36% SDS, 1.4 mg/ml proteinase K, 6% glycerol, 0.015% bromophenol blue followed by 15 min incubation at 37 °C. The
DNA products were analyzed by electrophoresis in 1.2% agarose gels in a buffer containing 40 mM Tris acetate, pH 5.5, 5 mM sodium acetate, and 1 mM MgCl2 at
3.5 V/cm for 2 h at 4 °C. The gels were dried on DE81 chromatography paper
(Whatman) and quantified using a Storm 840 PhosphorImager (GE Healthcare).
Gel retardation assay
The non-mobile 32P-labeled PX junction (oligos 174/175/176/181) (30 nM,
molecules) was incubated in binding buffer containing 25 mM Tris acetate (pH 7.5),
5mM magnesium acetate, 100 µg/ml BSA, 2 mM DTT, 2 mM ATP, and 10%
glycerol for 45 min at 4 °C. Then RAD54 or RAD54 mutants (at indicated
concentrations) were added to the reaction mixture followed by additional
incubation for 45 min. RAD54-DNA complexes were analyzed by electrophoresis
in 6% non-denaturing polyacrylamide gels (29:1) in 0.25x TBE buffer (22.5 mM
Tris borate pH 8.3 and 0.5 mM EDTA). The gels were dried on DE81
56 chromatography paper (Whatman) and the results were quantified using a Storm
840 PhosphorImager (GE Healthcare).
In vitro CDK2 phosphorylation assay
RAD54 or RAD541-142 (500 ng) was incubated in a 10 µl reaction mixture
containing 50 mM HEPES-KOH, pH 7.5, 10 mM MgCl2, 1 mM DTT, 30 µM ATP
and 83.5 nM [γ-32P] ATP (6000 mCi/mmole) with 500 ng of purified CDK2/CyclinA2
complexes for 30 min at 30 °C. The reactions were stopped by addition of EDTA to 25 mM and an equal volume of 2 x Laemmli buffer (20% glycerol, 0.3 M β-
mercaptoethanol, 4% SDS, 0.01% bromophenol blue, and 112 mM Tris HCl, pH
6.8) and incubation for 5 min at 75 °C. Proteins were analyzed by SDS-PAGE, and
gels were stained with Blue protein stain (Denville) as described by the
manufacturer. The gels were then dried on DE81 chromatography paper and
radioactive protein bands were quantified using a Storm 840 PhosphorImager (GE
Healthcare). To estimate the extent of RAD54 phosphorylation, we used
radioactive standards that were prepared by diluting [γ-32P] ATP stock 1,000, 5,000
and 10,000 times, and applying 1 µl of each dilution onto DE81 chromatography
paper that was exposed together with the gel containing phosphorylated RAD54.
57
D-loop formation
The nucleoprotein filaments were formed by incubating RAD51 protein with
32P-labeled ssDNA (#90, 90-mer) in buffer containing 35 mM Tris-HCl, pH 7.5, 2
mM ATP, 100 µg/ml BSA, 1 mM DTT, 20 mM KCl (from the protein stock), 1.5 mM
MgCl2, 0.5 mM CaCl2, and the ATP regenerating system (30 units/ml creatine
phosphokinase and 20 mM phosphocreatine for 30 min at 37 °C. The reaction was
transferred to 30 °C followed by addition of RAD54 and supercoiled pUC19 dsDNA
(50 µM nucleotide or 9.3 nM molecules) to initiate D-loop formation. Aliquots (10
µl) were withdrawn at indicated time points, and D-loops were deproteinized by
treatment with 5 µl of stop solution (1.36% SDS, 1.4 mg/ml proteinase K, 6%
glycerol, 0.015% bromophenol blue) for 15 min at 37 °C ; and analyzed by
electrophoresis in 1% agarose-TAE (40 mM Tris acetate, pH 8.0, and 1 mM EDTA)
gels. The gels were dried on DE81 chromatography paper and quantified using a
Storm 840 PhosphorImager (GE Healthcare).
ATPase Assay using Thin Layer Chromatography (TLC)
The phosphorylated or non-phosphorylated RAD54 protein (60 nM) was
incubated with PX-junction (10 nM, molecules) and 8 mM ATP, 20 nCi of [γ-
32P]ATP in 10 µl of reaction buffer containing 25 mM Tris acetate, pH 7.5, 2 mM
DTT, 100 μg/ml BSA, 11 mM magnesium acetate at 30 °C. The level of ATP
hydrolysis was determined using TLC on PEI-cellulose plates in running buffer
containing 1 M formic acid and 0.3 M LiCl. The products of ATP hydrolysis were quantified using a Storm 840 PhosphorImager (GE Healthcare).
58
RAD54 protein cross-linking
RAD54 or RAD54 mutants (1200 nM) were incubated in 10 µl reaction mixtures containing 20 mM HEPES-KOH, pH 7.5, 5 mM MgCl2, 5 mM EDTA, and
40 mM KCl with PX-junction (oligos 174/175/176/181) (0.4 µM, molecules) for 5
min at 25 °C. Followed by, the addition of bis-maleimidohexane (BMH) (Pierce) to
a final concentration of 25 µM. After a 10-min incubation at 25 °C, the reactions were quenched by the addition of β-mercaptoethanol to a final concentration of 1.1
M. The samples were analyzed by electrophoresis in 7.5% or 15% SDS-PAGE gels for RAD54 or RAD541-142 proteins, respectively. The proteins were visualized
by silver staining (Invitrogen).
2.4 RESULTS
2.4.1 The RAD54 N-terminal domain is essential for branch migration of
Holliday Junctions
We wished to explore the role of the N-terminal domain in the branch
migration activity of RAD54. We constructed a truncated RAD5496-747 mutant
devoid of the N-terminal 95 amino acid (aa) residues and tested its branch
migration activity using 32P-labeled partial Holliday junctions (PX) substrate (no.
71/169/170/171) that contains three dsDNA arms and one ssDNA arm (Figure
2.1A). Previously, we showed that the PX junction is a preferable substrate for
RAD54 branch migration (Bugreev et al., 2006b; Mazina et al., 2007). To reduce spontaneous branch migration, a single bp of heterology was introduced in one of
59 the four DNA arms, which during branch migration would create a single mismatched bp in each of the two branch migration products (Figure 2.1A). We found that the RAD5496-747 mutant has no detectable branch migration activity in a
wide range of tested protein concentrations (Figure 2.1B-E).
Since branch migration activity of RAD54 depends on ATP hydrolysis
(Bugreev et al., 2006b), we tested whether the loss of branch migration activity by
RAD5496-747 is due to disruption of its ATPase activity. Surprisingly, we found that
the RAD5496-747 has an approximately 2-2.5-fold greater ATPase activity than
RAD54 WT (Figure 2.1F). Thus, the N-terminal domain truncation specifically disrupts the branch migration, but not the ATPase activity of RAD54.
60
A
B
C
61 D
E
F
62
Figure 2.1 The RAD54 N-terminal domain is essential for branch migration, but not for ATP hydrolysis. A) The experimental scheme of the branch migration reaction. Shown is one bp heterology that was introduced into the PX junction (no. 71/169/170/171) to reduce the spontaneous branch migration. The shaded region denotes the heterologous DNA branch. The direction of branch migration is denoted by arrows. The asterisk indicates the 32P-label. The numbers correspond to oligonucleotides in Table 2.1. B) The kinetics of branch migration of PX-junction (10 nM, molecules) promoted by RAD54 (30 nM) or RAD5496-747 (30 nM). The branch migration products were analyzed by electrophoresis in 8% polyacrylamide gels. C) Data from B is plotted as a graph. D) The DNA products of branch migration reactions promoted by RAD54 or RAD5496-747 on PX-junction (no. 71/169/170/171) (10 nM, molecules) were analyzed by electrophoresis in 8% polyacrylamide gels. E) Data from D plotted as a graph. F) ATP hydrolysis by RAD54 (20 nM) and RAD5496-747 (20 nM) using supercoiled pUC19 as DNA substrate. Each experiment was repeated three times. Error bars represent the standard error of the mean (S.E.)
2.4.2 RAD5496-747 retains DNA translocation activity
We wanted to determine the specific role of the N-terminal domain in
RAD54-promoted branch migration. At minimum, a branch migration protein must translocate along the DNA axis in an ATPase-dependent manner. We tested whether RAD5496-747 retains DNA translocation activity using a triple-helix displacement assay (Figure 2.2A) (Deakyne et al., 2013; Firman and Szczelkun,
2000; Jaskelioff et al., 2003). The triple-helix was formed under slightly acidic
conditions, pH 5.5, by annealing a pyrimidine-rich triplex forming oligonucleotide
(TFO, 22 mer) to a purine-rich sequence in pMJ5 plasmid DNA through Hoogsteen base pairing. The triple-helix is stable at neutral pH, however, once disrupted by a
DNA translocating protein, it will not re-form. We found that the RAD5496-747 not only retained DNA translocation activity but it was increased approximately 2-2.5-
fold relative to that of RAD54 WT (Figure 2.2B-C). Thus, the DNA translocation
63
activity of RAD5496-747 was increased in parallel with an increase in the ATPase activity, but in a sharp contrast with the loss of the branch migration activity (Figure
2.1).
A
B
64
C
D
65
E
F
66
Figure 2.2 The 95 aa N-terminal truncation enhances DNA translocation activity of RAD54. A) The experimental scheme of the triple-helix displacement assay. The zig-zag line indicates triple-helix forming oligonucleotide (TFO) that is paired to the linearized pMJ5 plasmid. The asterisk indicates the 32P-label. B) The kinetics of triple-helix (0.5 nM, molecules) displacement by RAD54 (5 nM) or RAD5496-747 (5 nM) was analyzed by electrophoresis in 1.2 % agarose gels. C) Data from B plotted as a graph. D) The ATPase activity of RAD54 (60 nM) and RAD5496-747 (60 nM) was measured in presence of various DNA substrates; ssDNA (no. 2), dsDNA (no. 1/2), PX-junction (no.174/175/176/181), and Holliday junction (no.174/175/176/177) (20 nM, molecules) at 30 °C. E) DNA binding 32 preferences for RAD5496-747. RAD5496-747 (150 nM) was incubated with P-labeled non-mobile PX-junction (30 nM, no. 174/175/176/181) in the presence of the indicated concentrations of unlabeled DNA competitors (ssDNA, dsDNA, PX- junction, X-junction). The complexes were analyzed by EMSA. F) Data from E plotted as a graph. Each experiment was repeated three times. Error bars represent the S.E.
2.4.3 The N-terminal 95 aa truncation changes DNA binding preferences of
RAD54
Previously, we showed that RAD54 has a strong binding preference for
branched DNA substrates, including PX-junction and HJ-junctions (Bugreev et al.,
2006b; Mazina et al., 2007). Here, we tested DNA binding preferences of RAD5496-
747 using the DNA-dependent ATPase as a read-out and electrophoretic mobility
shift assays (EMSA). We found that the ATPase activity of RAD5496-747, similar to
RAD54 WT, was stimulated greater by branched DNA substrates than by linear dsDNA (Figure 2.2D). However, RAD5496-747 had a similar preference for PX and
Holliday junction substrate, unlike RAD54 WT, which had a higher preference for
PX over Holliday junction substrate.
67
We also tested DNA binding specificity of RAD5496-747 using EMSA
32 (Bugreev et al., 2006b). RAD5496-747 complexes with P-labeled PX-junction (no.
174/175/176/181) were formed in the presence of the increasing concentrations of unlabeled DNA competitors of different structures and analyzed by gel- electrophoresis. The concentration of DNA competitors required to reduce the
32 complex formation between RAD5496-747 and the P-labeled PX junction by 50%
were approximately 60 nM for PX-junction (no. 174/175/176/181), 130 nM for
Holliday junction (no. 174/175/176/177), 1400 nM for dsDNA (no. 1/2), and 3000
nM for ssDNA (no. 2) (Figure 2.2E-F). These results demonstrate that while the N-
terminal 95 aa truncation does not disrupt the ability of RAD54 to interact
specifically with Holliday junction-like DNA substrates, it lowers the relative
preference for PX vs Holliday junction substrates (~2-fold) in comparison to RAD54
WT (~5-fold) (Bugreev et al., 2006b). These data together with the results of the
ATPase assay (Figure 2.2D) indicate a change in DNA binding specificity of the N-
terminal truncated RAD5496-747 compared with RAD54 WT.
2.4.4 The RAD54 N-terminal domain binds preferentially to branched DNA
substrates
In addition to the DNA binding site in the core ATPase/DNA binding domain,
RAD54 has a secondary DNA binding site in the N-terminal domain (Wright and
Heyer, 2014). To assess a possible role of this site in RAD54 branch migration of
Holliday junctions, we expressed the RAD541-142 N-terminal domain in E. coli
(Golub et al., 1997), purified it, and studied its DNA binding properties. First, using
68
EMSA we found that RAD541-142 binds DNA and has even stronger binding preference for branched DNA than RAD5496-747, as no significant disruption of the
32 RAD541-142 complex with P-labelled PX-junctions (no. 174/175/176/181) was observed in the presence of a 150-200 fold excess of cold ss- or ds-DNA (no. 2 or
no. 1/2) competitor (Figure 2.3A-B). RAD541-142 also has a strong, approximately
6-fold, preference for PX-junction over HJ-junction substrates, which is stronger than that of RAD5496-747 (Figure 2.2F), but similar to RAD54 WT (Bugreev et al.,
2006b). These data suggest that the N-terminal domain DNA binding site may play an important role in determining the preferential binding of RAD54 to Holliday junction-like structures. The N-terminal 95 aa deletion may partially disrupt this site changing the DNA binding specificity of RAD54.
A
69
B
Figure 2.3 The DNA binding preference of RAD541-142. A) DNA binding 32 preferences for RAD541-142. RAD541-142 (300 nM) was incubated with P-labeled non-mobile PX-junction (30 nM, no. 174/175/176/181) in the presence of the indicated concentrations of unlabeled DNA competitors (ssDNA, dsDNA, PX- junction, X-junction). The complexes were analyzed by EMSA. B) Data from A plotted as a graph. Each experiment was repeated three times. Error bars represent the S.E.
2.4.5 Mutations that separately inhibit N-terminal domain oligomerization or
DNA binding
In order to identify the specific aa residues essential for DNA binding of
RAD541-142, we generated 3 mutants by substituting stretches of basic aa residues
KRK12-14AAA RKRK33- with alanine residues; RAD541-142 (aa residues 12-14), RAD541-142
36AAAA RK52-53AA (aa residues 33-36), and RAD541-142 (aa residues 52-53), (Table
T31E S49E 2.2). We also constructed RAD541-142 and RAD541-142 phosphomimetic
70 mutants, since RAD54 was found to be phosphorylated at S49 HeLa cells
(Kettenbach et al., 2011). This site is consensus sequence for CDK2 phosphorylation. Further analysis showed that there are two CDK2 phosphorylation consensus sites located in the RAD54 N-terminal domain, at positions 30-34 (VTPRK) and 48-52 (LSPFR). We wanted to test the effect of
CDK2 phosphorylation at these sites on DNA binding by the RAD54 N-terminal
KRK12- domain. All RAD54 N-terminal domain mutant proteins, except RAD541-142
14AAA , which failed to express under various tested growth conditions, were purified
from E.coli, and examined for binding to PX-junction (no. 174/175/176/181) using
EMSA.
RK52-53AA RAD541-142 mutant bound DNA similar to the non-mutated RAD541-
142 that formed a series of distinct complexes starting at 0.1 µM protein
concentration (Figure 2.4A, lanes 8-12) (data not shown). In contrast, RAD541-
RKRK33-36AAAA 142 showed only a weak binding to PX-junction. No distinct complexes
were observed on the gel, however, smearing observed at the highest protein
concentration tested (0.5 µM) likely indicated the formation of unstable DNA-
protein complexes prone to dissociation during gel electrophoresis (Figure 2.4A,
lanes 1-6).
The S49E mutation reduced DNA binding of RAD541-142, specifically, the formation of large nucleoprotein complexes; whereas formation of small fast migrating complexes was not significantly affected (Figure 2.4A, lanes 13-17). In contrast, the T31E mutation had no significant effect on DNA binding by RAD541-
142 (Figure 2.4B, lanes 2-6). Thus, both the RKRK33-36AAAA and S49E mutations
71
reduced DNA binding of RAD541-142, but while the former mutation has an overall
destabilizing effect on RAD541-142 DNA binding, the latter one specifically disrupted
the formation of large nucleoprotein complexes.
In solution, RAD54 is present in a monomeric form but multimerizes in the
presence of DNA (Petukhova et al., 1999). Previously, we suggested that DNA-
dependent RAD54 oligomerization is necessary for branch migration (Mazina et
al., 2007). To test if either of the two N-terminal domain mutations, RKRK33-
36AAAA or S49E, affect the RAD54 oligomerization, we performed crosslinking
experiments using BMH agent that links two cysteine thiol groups separated by a
S49E RKRK33-36AAAA distance no more than 13 Å. RAD541-142, RAD541-142 or RAD541-142 proteins were incubated with minimal DNA flap substrate (no. 244/249/250) containing two 15-bp dsDNA arms and one 45-nt ssDNA arm, the smallest DNA substrate that is still capable to efficiently stimulate the ATPase activity of RAD54
(Mazina et al., 2007) followed by BMH crosslinking. The products of the protein crosslinking were analyzed on an SDS-polyacrylamide gel. Untreated RAD541-142,
S49E RKRK33-36AAAA RAD541-142 , and RAD541-142 migrated in accord with the predicted
size (~16 kDa) (Figure 2.5, lanes 2, 5, and 9). In the absence of DNA, BMH
treatment gave rise to a small additional band of ~36 kDa, consistent with predicted
dimer size (32 kDa) (Figure 2.5, lanes 3, 6, and 8). In the presence of DNA, BMH
crosslinking resulted in a larger fraction of dimers (Figure 2.5, lanes 4, 7, and 10).
RKRK33-36AAAA In the case of RAD541-142 and RAD541-142 , large oligomers were also
observed, some of them were unable to enter the gel (Figure 2.5, lanes 4 and 10).
Apparently, BMH crosslinking helped to visualize intrinsically unstable
72
RKRK33-36AAAA nucleoprotein complexes formed by RAD541-142 (Figure 2.4A; lanes 1-
6). In contrast, higher order oligomers were nearly completely absent in RAD541-
S49E 142 (Figure 2.5, lane 7).
Thus, RAD54S49E and RAD54RKRK33-36AAAA mutations differently affect the interaction of the RAD54 N-terminal domain with PX-junctions. RAD54S49E did not significantly disrupt the formation of fast migrating complexes in EMSA and formation of protein dimers in crosslinking experiments. However, this mutation
decreased dramatically formation of large protein oligomers. To the contrary,
RAD54RKRK33-36AAAA did not affect DNA-dependent protein oligomerization but
significantly reduced the formation of stable protein-DNA complexes visualized by
EMSA.
A
73
B
C
Figure 2.4 Mutations in RAD541-142 disrupt its DNA binding. A) The effect of the S49E and RKRK33-36AAAA mutations on RAD541-142 binding to PX-junction (30 nM, no. 174/175/176/181) was analyzed by EMSA in a 6% polyacrylamide gel. B) The effect of the S49E and T31E mutations on RAD541-142 binding to PX- junction (30 nM, no. 174/175/176/181) was analyzed by EMSA in a 6% polyacrylamide gel. C) Data from A & B plotted as a graph. Each experiment was repeated three times. Error bars represent the S.E.
74
Figure 2.5 The S49E, but not the RKRK33-36AAAA, mutation inhibits DNA- dependent oligomerization of RAD541-142. The proteins (1200 nM) were incubated with or without the minimal flap DNA (no. 244/249/250) (400 nM) in the presence or absence of BMH (25 µM) and analyzed in a 15% SDS-PAGE. Arrows indicate migration of the monomeric, dimeric, and oligomeric protein products. The molecular weight standards (Precision Plus; Bio-Rad) are shown. Each experiment was repeated three times. Error bars represent the S.E.
2.4.6 The N-terminal domain mutations inhibit RAD54 branch migration
Next, we wished to test the effect of the RKRK33-36AAAA and S49E
mutations on the branch migration activity of RAD54. We constructed full length
RAD54 carrying these N-terminal domain mutations with a sumo-His tag, expressed them in E.coli and purified. In control experiments, we demonstrated that the ATPase and branch migration activities of RAD54 WT with this tag purified
75 from E.coli were similar to that of RAD54 purified from insect cells (Figure 2.6A,
B). Using PX-junctions (no. 71/169/170/171) as a substrate, we found that the branch migration activities of RAD54RKRK33-36AAAA and RAD54S49E were reduced
(Figure 2.7A). This inhibition could not be due to a decrease in the ATPase activity which was even slightly elevated in both mutants relative to RAD54 WT (Figure
2.7B). Thus, the mutations that either disrupted DNA binding of the N-terminal domain or inhibited its oligomerization cause inhibition of RAD54 branch migration activity.
Figure 2.6 Comparison of activities of RAD54 purified from E.coli or insect cells. A) The kinetics of branch migration of PX-junction (10 nM, molecules) promoted by RAD54 WT (30 nM) purified from insect cells or from E.coli. The branch migration products were analyzed by electrophoresis in 8% polyacrylamide gels. B) ATP hydrolysis by RAD54 WT (20 nM) purified form insect cells or from E.coli using supercoiled pUC19 as DNA substrate. Each experiment was repeated three times. Error bars represent the S.E.
76
A
B
77
Figure 2.7 The S49E and RKRK33-36AAAA mutations inhibit branch migration, but not ATP hydrolysis. A) The effect of the S49E and RKRK33- 36AAAA single and double mutations on the kinetics of branch migration of PX- junction (32 nM, no. 71/169/170/171) by RAD54 (60 nM). B) The effect of the S49E and RKRK33-36AAAA mutations on ATP hydrolysis by RAD54 (20 nM) using supercoiled pUC19 as DNA substrate. Each experiment was repeated three times. Error bars represent the S.E.
If two mutations inhibit branch migration by different mechanisms, one could expect that their combination would inhibit branch migration stronger than any of the mutations individually. Indeed, we found that in the RAD54RKRK33-36AAAA, S49E double mutant, the inhibition of branch migration activity was stronger than in either of the single mutants (Figure 2.7A, B) consistent with the different mechanisms of inhibition caused by these mutations: DNA binding vs protein oligomerization.
2.4.7 N-terminal domain phosphorylation by CDK2 inhibits RAD54 branch migration but not its stimulation of RAD51 strand exchange
Because RAD54 oligomerization, which is required for branch migration, is disrupted by S49E phosphomimetic mutation, we wanted to examine directly the effect of the N-terminal domain phosphorylation by CDK2 on the RAD54 branch migration activity. We first tested whether CDK2/cyclinA2 can phosphorylate
RAD54 and the RAD541-142 in vitro. We observed that both proteins were phosphorylated with similar efficiency, consistent with the position of two CDK2 consensuses in the RAD54 N-terminal domain (Figure 2.8A). When we used the
RAD54S49E and RAD54T31E, S49E mutants as substrates for CDK2/cyclinA2, the level
of phosphorylation was reduced to 65% and 15% for the single and double mutant,
78 respectively, indicating that T31 and S49 are the major phosphorylation sites in
RAD54 (Figure 2.8B). Next, we tested the effect of CDK2 phosphorylation on the branch migration activity of RAD54. We found that phosphorylation reduced
RAD54 branch migration activity (Figure 2.9A). In contrast, we did not observe any effect of CDK2 phosphorylation on the ATPase activity of RAD54 (Figure 2.9B).
We also found that CDK2 phosphorylation did not inhibit RAD54 ability to stimulate
DNA strand exchange activity of RAD51 (Figure 2.10A and 2.10B). These data suggest that CDK2 may specifically regulate RAD54 branch migration activity through phosphorylation of the N-terminal domain site.
A
79
B
Figure 2.8 CDK2 phosphorylates the N-terminal region of RAD54 in vitro. A) Left panel, Autoradiograph showing in vitro phosphorylation of RAD54 (500 ng) 32 and RAD541-142 (500 ng) in the presence of [γ- P] ATP with purified CDK2/cyclin complex. Right panel, Denville blue protein staining of the gel in the left panel. B) Left panel, Autoradiograph showing in vitro phosphorylation of RAD54 WT (500 ng), RAD54S49E (500 ng), and RAD54T31E, S49E (500 ng) with CDK2/cyclin complex using [γ-32P] ATP. Right panel, Denville blue protein staining of the gel shown in the left panel.
80
A
B
Figure 2.9 Effect of CDK2 phosphorylation on the RAD54 branch migration and ATPase activity. A) The kinetics of branch migration of PX-junction (no. 71/169/170/171) (32 nM) promoted by RAD54 (60 nM) or phosphorylated RAD54 (60 nM). B) The kinetics of ATP hydrolyzed by RAD54 (60 nM) or phosphorylated RAD54 (60 nM) in presence of PX-junction (no. 174/175/176/181) (60 nM, molecule). The ATP hydrolysis was measured using Thin-Layer chromatography.
81
A
B
C
82
Figure 2.10 Effect of phosphorylated RAD54 on strand exchange activity of RAD51. A) The experimental scheme of the D-loop reaction. The nucleoprotein filaments were formed between RAD51 and ssDNA at 37°C for 30 min. The reactions were then moved to 30°C. Then RAD54 was added followed by addition of pUC19 dsDNA substrate to initiate the reaction. B) The effect of RAD54 (50 nM) or phosphomimetic RAD54S49E (50 nM) on D-loop formation by RAD51 (1.25 µM) between 32P-labeled ssDNA (oligo 90, 2.4 µM nts) and pUC19 (50 µM, nts). The D-loops were analyzed by electrophoresis in 1% agarose gel. C) Data from B plotted as a graph. Each experiment was repeated three times. Error bars represent the S.E.
2.5 DISCUSSION
RAD54 is important homologous recombination protein evolutionarily
conserved from low to higher eukaryotes (Mazin et al., 2010). In vitro, it possesses several activities including stimulation of the RAD51 DNA strand exchange activity and remodeling different dsDNA-protein complexes (Alexeev et al., 2003; Mazina and Mazin, 2004; Petukhova et al., 1998). However, RAD54 stands out as the most proficient branch migration protein in eukaryotes known so far (Bugreev et al.,
2006b; Mazin et al., 2010; Mazina et al., 2012). Moreover, RAD54 is the only
known eukaryotic protein on par with E.coli RuvAB that can bypass large regions
of DNA heterology (~100 bp) during branch migration of Holliday junctions (Mazin
et al., 2010; Mazina et al., 2012). This property is remarkable because, in contrast
to most other branch migration proteins, including RuvAB or BLM, RAD54 lacks
canonical DNA helicase activity. However, the mechanism of RAD54 branch
migration remains to be elucidated.
In spite of significant structural diversity, all branch migration proteins show
high affinity to Holliday junction or Holliday junction-like structures and an ability to
83 translocate on DNA in an ATPase dependent manner. In the prototypic RuvAB complex, these two functions are segregated between RuvA and RuvB proteins, which act as the structure-specific DNA-binding protein and the ATPase motor protein, respectively (West, 1997). However, RAD54 and other known branch
migration proteins including bacterial RecG and eukaryotic RecQ family helicases,
BLM, RECQ1, and WRN, combine these two important activities in a single
polypeptide.
Our current data demonstrates that the N-terminal domain is essential for
RAD54 branch migration activity. The RAD5496-747 mutant that lacks a significant
portion of the N-terminal domain is devoid of branch migration activity nearly
completely, while still proficient in DNA translocation and in ATP hydrolysis. We
found that the RAD54 N-terminal domain promotes specific binding of RAD54 to
Holliday junction substrates. The RAD541-142 has the preference for Holliday
junction-like DNA substrates as strong as the full length RAD54. Replacement of
basic a.a. residues 33-36 by alanines (RKRK33-36AAAA) diminished DNA binding
by the RAD54 N-terminal domain indicating the tentative position of the N-terminal
domain DNA binding site. Interestingly, RAD5496-747 retains the ability to recognize
Holliday junction-like structures, albeit with an altered specificity relative to different
types of branched DNA molecules. This result indicates that the N-terminal domain
area between 96 and 142 aa residues may also contribute to the recognition of
Holliday junction-like structures. Although the RAD54 N-terminal shows no
structural homology outside of the RAD54 protein family it may represent a functional analog of the N-terminal domain of RecG or of the RecQ helicases CTD
84
(RQC) that are responsible for specific binding to branched DNA structures (Kim et al., 2013; Kitano et al., 2010; Pike et al., 2009 ; Singleton et al., 2001).
Furthermore, deletion the RAD54 N-terminal domain, similar to deletion of the BLM
HRDC domain adjacent to RQC (Swan et al., 2014), increased the ATPase activity of the enzyme. It is thought that HRDC may suppress the BLM ATPase activity through direct interaction with the ATPase core.
The proteolytic cleavage analysis shows that in S. cerevisiae, the N-terminal domain of Rad54 is unstructured in solution (Raschle et al., 2004). These data are consistent with the structural flexibility of the RAD54 N-terminal domain, which enables its interaction with various partners, like RAD51, CDK2, or DNA (Golub et al., 1997; Kettenbach et al., 2011; Raschle et al., 2004; Wright and Heyer, 2014).
Our current data further emphasize the structural flexibility of the RAD54 N- terminal domain by demonstrating a coupling between its binding to DNA and oligomerization. Previously, we showed that RAD54 forms oligomeric structures on Holliday junction substrates during branch migration (Mazina et al., 2007), the property shared with other branch migration proteins including BLM and RuvAB
(Mazina et al., 2012; West, 1997). However, the structural determinant of RAD54 oligomerization remained unknown. Our current data show that RAD54 N-terminal domain is responsible for DNA-dependent RAD54 oligomerization. We found that the N-terminal domain alone is both necessary and sufficient for protein oligomerization and that the N-terminal truncation, significantly reduced the formation of RAD5496-747 oligomers in the presence of DNA. Moreover, we
identified N-terminal domain mutations that separately affect DNA binding and
85 protein oligomerization. Thus, RKRK33-36AAAA mutation decreases DNA binding
of the RAD541-142 to DNA significantly. However, even this residual DNA binding
appeared to be sufficient to induce protein oligomerization identified in BMH cross-
linking experiments. In contrast, S49E mutation only weakly inhibits initial binding
of RAD541-142 to DNA but disrupts the formation of large oligomeric protein complexes visualized by BMH cross-linking. In full-size RAD54, each of these mutations inhibited branch migration and their combination increased the inhibition.
Taken together, these data led us to suggest a two-step process of formation of the active RAD54 branch migration complex. First, the RAD54 N- terminal domain specifically targets RAD54 to Holliday junction. In the presence of
Mg2+ (>100 µM), Holliday junction exists in stacked conformation that is refractory
to branch migration (Mazina et al., 2007). Based on structural data, it was recently
proposed that binding of the BLM RQC domain induces structural transition of
Holliday junction into open conformation that is proficient in branch migration (Kim
et al., 2013). We suggest that binding of the RAD54 N-terminal domain may have
a similar effect on the Holliday junction conformation. Second, binding of the
RAD54 N-terminal domain to DNA enables RAD54 oligomerization that is required
for branch migration (Mazina et al., 2007).
A phosphoproteomics study identified RAD54 phosphorylation at S49
(Kettenbach et al., 2011). Two CDK2 consensus sites are located in the RAD54
N-terminal domain, with putative phosphorylation sites at T31 and S49. Our in vitro
CDK2 phosphorylation data are consistent, T31 and S49 as major phosphorylation
86 sites in RAD54, as T31E, S49E double mutations significantly reduced CDK2
phosphorylation of RAD54. We found that S49E, but not T31E phosphomimetic
mutation, inhibits RAD54 N-terminal domain oligomerization. Being incorporated
in the full-size RAD54, this mutation inhibited the RAD54 branch migration activity
consistent with the important role of RAD54 oligomerization for branch migration.
The effect of the S49E mutation is specific for branch migration, as it has no
significant effect on stimulation of RAD51-promoted DNA strand exchange by
RAD54 indicating that RAD54 oligomerization is not required for stimulation of
DNA strand exchange promoted by RAD51. In vitro CDK2 phosphorylation of
RAD54 also inhibited RAD54 branch migration showing even stronger effect than the phosphomimetic S49E mutation.
Previously, we showed that RAD54 branch migration activity causes dissociation of D-loops, the product of ssDNA invasion into homologous DNA- template during double-strand break repair (Bugreev et al., 2007). This step of double-strand break repair, however, needs to be precisely controlled, as premature disruption of D-loops prior to their extension by DNA polymerase may abort double-strand break repair. CDK2-dependent inhibition of RAD54 branch migration may help to delay D-loop disruption until the polymerization step is completed (Figure 2.11). Recently, it was shown that Nek1 phosphorylates RAD54 at S572 in late G2 phase to modulate its interaction with RAD51 (Spies et al.,
2016). Thus, multiple mechanisms are in place to control different RAD54 activities in the cell.
87
Figure 2.11 CDK2-dependent RAD54 regulation. The initial steps of homologous recombination involve, 5’ to 3’ exonucleolytic processing of double- strand break ends producing 3’‐ssDNA overhangs, the formation of RAD51‐ssDNA nucleoprotein filaments, search for homology and strand invasion into the homologous duplex DNA‐template leading to the formation of displacement loops (D‐loop). RAD54 or pRAD54 both can stimulate the strand exchange activity of
88
RAD51. RAD54, through its branch migration activity, can also dissociate D-loops prior to the extension of the invading DNA strand disrupting the double-strand break repair process. pRAD54 is unable to oligomerize, thus inhibiting branch migration activity of RAD54 which provides sufficient time for the extension of invading strand. During later stages of homologous recombination, RAD54 needs to be in dephosphorylated form so that branch migration could occur to complete the repair process. Thus, phosphorylation by CDK2 modulates activities of RAD54 during homologous recombination.
In summary, we show that RAD54 N-terminal domain plays a pivotal role in the branch migration activity by specific binding to Holliday junctions and promoting
DNA-dependent oligomerization. Furthermore, the RAD54 N-terminal domain, being a target for CDK2 phosphorylation, may play a role of the hub that controls
RAD54 activities in the cell.
89
Characterization of RAD54 promoted annealing activity
3.1 INTRODUCTION
RAD54 belongs to the SWI2/SNF2 family of helicase-like proteins and is
conserved in all eukaryotes. The members of this family of helicase-like proteins
are known for their chromatin remodeling activity. The helicase-like proteins do not
have DNA strand separation activity like canonical helicases, however, they use
DNA-dependent ATP hydrolysis to translocate along the DNA generating positive
supercoils in the DNA ahead and negative supercoils behind the protein. This
allows the DNA strands to transiently “breathe”. RAD54 promotes chromatin
remodeling and displacement of proteins from double-stranded DNA. It binds
Holliday junctions and drives their branch migration (Bugreev et al., 2006b).
RAD54 interacts with Mus81-Eme1 (Mms4), a structure-specific endonuclease,
stimulating its DNA cleavage activity (Matulova et al., 2009; Mazina and Mazin,
2008). Thus, RAD54 is a multifunctional protein performing functions throughout
homologous recombination pathway and linking the whole process together
(Heyer et al., 2006; San Filippo et al., 2008; Symington and Heyer, 2006; Tan et
al., 2003).
RAD54 is a member of Superfamily (SF) 2 of helicases (Troelstra et al.,
1992) and possesses seven classical “signature” motifs: I, Ia, II, III, IV, V, and VI,
a characteristic of SF2 protein members (Nimonkar et al., 2007) plus several more
recently identified motifs, like TxGx between motif Ia and motif II that is thought to
play a role in oligonucleotide binding. The seven conserved motifs line the two
90
RecA-like motor domains, constituting the “core” or the minimal translocation motor responsible for the conversion of energy from ATP hydrolysis to the mechanical energy needed for translocation (Singleton et al., 2007).
RAD54 sequencing identified it as a member of the group of proteins involved in translocation along nucleic acids that are broadly defined as helicases
(Gorbalenya and Koonin, 1993; Troelstra et al., 1992). The common feature of these proteins is that they couple ATP hydrolysis to directional movement along nucleic acids, which may or may not result in strand separation of nucleic acids.
Members of this group have a role in almost every cellular process from DNA replication and repair, transcription, translation, splicing, and nuclear transport
(Singleton et al., 2007).
Helicases are molecular motors coupling the energy of nucleoside triphosphate hydrolysis to the unwinding and remodeling of structured DNA or RNA
(Lohman and Bjornson, 1996; Patel and Donmez, 2006; Singleton et al., 2007). In higher organisms, approximately 1% of the genes of eukaryotic genomes apparently encode RNA or DNA helicases. Helicases can be classified as DNA or
RNA helicases, depending upon their substrates. Although some helicases can function on both DNA and RNA molecules (Pyle, 2008). DNA helicases function in a variety of DNA metabolic processes, including unwinding duplex or alternative
DNA structures (triplex, G-quadruplex), displacing protein bound to DNA, and chromatin remodeling (Bernstein et al., 2010; Dillingham, 2011; Wu et al., 2009).
Traditionally, helicases have the ability to unwind double-stranded DNA or RNA in an ATP-dependent manner. There is, however, increasing evidence that some
91 helicases can anneal complementary strands of DNA or RNA molecules in the presence or absence of nucleoside triphosphate. The mechanism of this strand annealing activity of helicases and its biological relevance is not yet completely understood.
Several members of the RecQ family of helicases have been found to possess annealing activity. There are five RecQ homologs in humans, and mutations in three of these genes (BLM, WRN, and RECQ4) are associated with
Bloom’s, Werner, and Rothmund-Thomson syndromes, respectively. The mutations in RECQ5 have not been linked to any diseases, however, recq5−/− mice display genomic instability and are prone to cancer (Hu et al., 2005; Hu et al.,
2007). A single nucleotide polymorphism in RECQ1 is shown to correlate with decreased survival of pancreatic cancer patients (Li et al., 2006a; Li et al., 2006b).
Several DNA branch migration proteins, BLM, WRN, and RECQ1, have been shown to possess annealing activity. Bloom’s syndrome helicase, BLM, has been to carry out annealing of two complementary DNA molecules (Cheok et al.,
2005; Machwe et al., 2005). Furthermore, it was shown that N-terminus of
BLM/Sgs-1 was responsible for the annealing activity observed in vitro (Chen and
Brill, 2010). The WRN helicase is also able to carry out annealing of complementary DNA molecules (Machwe et al., 2005) and this activity was mapped to the amino-acids 1072-1150 in the C-terminal region (Muftuoglu et al.,
2008). Annealing activity has also reported for the RECQ1 protein (Sharma et al.,
2005). RECQ1 efficiently promotes strand annealing as a higher order oligomer
(pentamer or hexamer), while smaller oligomeric states (dimer or monomer) act to
92 unwind duplex DNA (Muzzolini et al., 2007). Therefore, different oligomeric states of RECQ1, modulated by binding of ssDNA and ATP, are associated with its strand annealing or unwinding activity.
Unlike other branch migration proteins, annealing activity has not been reported for RAD54 protein. Recently, a second DNA-binding site was reported in the N-terminal domain of RAD54 (Wright and Heyer, 2014). Two DNA binding sites have also been reported for RAD51 and RAD52 proteins that carry out annealing and strand exchange between two DNA molecules (Khade and Sugiyama, 2016).
Therefore, the presence of two DNA binding sites in RAD54 may suggest the ability to anneal two complementary DNA molecules like other branch migration proteins.
Here, we show that RAD54 protein possesses efficient annealing activity.
The N-terminal truncated protein, RAD5496-747, had significantly reduced annealing
activity, and RAD541-142 did not promote ssDNA annealing. Therefore, the second
DNA binding site in the N-terminal region of RAD54 is essential but not sufficient to carry out annealing indicating the requirement for both DNA binding sites for annealing activity. Disrupting the DNA-dependent oligomerization of the N-terminal domain using S49E mutant, had no effect on the ability of RAD54 to promote annealing. However, RAD54RKRK33-36AAAA mutant showed significantly lower
annealing activity consistent with the requirement of two DNA binding sites for annealing activity.
93
3.2 MATERIALS AND METHODS
Proteins and DNA
All oligonucleotides used in this study (Table 3.1) were purchased from IDT
Inc and purified using denaturing 6-10% PAGE. To prepare oligonucleotide dsDNA
substrates, complementary ssDNA oligonucleotides were annealed as described
(Rossi et al., 2010) and stored at -20 °C. Oligonucleotides were labeled using [γ-
32P] ATP and T4 polynucleotide kinase. T4 polynucleotide kinase and restriction endonucleases were purchased from New England Biolabs. PreScission™
Protease was purchased from GE Healthcare Life Sciences.
Table 3.1. Sequences of the oligonucleotides used in this study *
Length in Number Sequence, 5’3’ nucleotides
GTC GAC GAC GTC TGA GTA CTC ATC TAG TGT GAC ATC #64 48 ATC GCA TCG AGA
TCT CGA TGC GAT GAT GTC ACA CTA GAT GAG TAC TCA #65 48 GAC GTC GTC GAC
Purification of RAD54 and N-terminal truncated mutants from insect cells
Human RAD54 and RAD5496-747 were purified from Sf21 cells using
baculovirus expression vector as described previously (Mazina and Mazin, 2004)
with alterations indicated below. The purification scheme included a Glutathione
94
S-transferase (GST) affinity column, followed by a Superdex 200 column, and then a Resource S column. In this work, we used RAD54 constructs containing an amino-terminal GST affinity tag separated from the RAD54 gene by a PreScission
Protease™ recognition sequence (L-E-V-L-F-Q-G-P). Following fractionation on the Superdex 200 column, the pool containing RAD54 was incubated with
PreScission Protease (GE Healthcare) (10 U of PreScission Protease per 100 mg of RAD54) for 4 h at 4 °C. The sample was diluted to 100 mM KCl and loaded on a 1 ml Resource S column. The fractions containing RAD54 or RAD54 mutants were analyzed for nuclease contamination, pooled, and stored in small aliquots at
-80 °C. The protein appeared nearly homogeneous in a Coomassie-stained SDS- polyacrylamide gel.
Expression and purification of RAD54, and RAD54 mutant proteins from
E.coli
Human RAD54, RAD54T31E, RAD54S49E, RAD541-142 (N-terminal 142 aa
RAD54 fragment), RAD541-142 T31E, RAD54 1-142 S49E, and RAD54RKRK4A, with
Sumo-His affinity tag, were cloned in the pETHSUL vector (Weeks et al., 2007) and expressed in E. coli Rosetta 2 cells. The cDNA sequence used for cloning was amplified using a pFastBac-HTc vector containing RAD54 cDNA sequence as template and DNA primers listed in Supplemental Table 2. The cloning was performed using Ligation independent cloning protocol as described previously
(Weeks et al., 2007). All RAD54 mutants were generated using QuickChange site directed mutagenesis kit (Agilent Technologies) using primers listed in
95
Supplemental Table 2. Rosetta 2 cells were transformed with the recombinant plasmids and cultured at 37 °C to an O.D.600 of approximately 0.4. The cultures
were transferred to 16 °C and allowed to grow until they reach an O.D.600 of 0.6-
0.8, following which the protein expression was induced with 0.1 mM isopropyl-1- thio-β-D-galactopyranoside for 20 h. Cells were harvested by centrifugation (7000 x g) and stored at -80oC. All purification steps were carried out at 4 °C. Cells (10
g) were thawed and resuspended in ten volumes of lysis buffer (20 mM KH2PO4 pH 7.5, 200 mM KCl, 10% Sucrose, 10 mM 2-mercaptoethanol) supplemented with
EDTA-free protease inhibitor cocktail tablets (Roche Applied Science). The cells were lysed by passing their suspension twice through an Emulsiflex C-5 (Avestin) at 15, 000 – 20, 000 psi. The crude extract was clarified by centrifugation (185,
000 X g for 60 min) and passed through 0.45 µm filter. The filtrate was loaded onto a 5 ml HiTrap Ni2+ column (GE Healthcare). The column was washed extensively
with buffer A (20 mM KH2PO4 pH 7.5, 500 mM KCl, 10% glycerol, 10 mM 2- mercaptoethanol) supplemented with 50 mM Imidazole. RAD54 protein was eluted
using buffer A supplemented with 500 mM Imidazole and the eluate was
supplemented with 2 mM EDTA. Sumo-His tags were cleaved off RAD54
constructs by adding Sumo hydrolase (dtUD1) (200 µg) to the eluate followed by
incubation for 4 h at 4 °C. The cleaved RAD54 was further fractionated on a
Superdex-200 column (58 ml) equilibrated with buffer A. The fractions containing
RAD54 were pooled, diluted 5 times using buffer B (20 mM KH2PO4 pH 7.5, 10%
glycerol, 10 mM 2-mercaptoethanol) and loaded on to 1 ml Resource S column
(GE Healthcare), equilibrated with buffer B supplemented with 100 mM KCl. The
96 protein was eluted with a 20 ml gradient of KCl (100-450 mM) in buffer B. The fractions containing RAD54 were analyzed for nuclease contamination, pooled, and stored in small aliquots at -80 °C. The protein appeared nearly homogeneous in a Coomassie-stained SDS-polyacrylamide gel.
The RAD541-142 was purified as above except that a 58-ml Sephacryl S-200
column (GE Healthcare) was used in place of Superdex-200.
DNA strand annealing assay
The annealing reactions were carried out using two complementary ssDNA
oligonucleotides (# 64 and #65, 48-mer each), in which one was 32P-labeled at the
5’-end. The non-labeled oligonucleotide (5 nM, molecules) was incubated in a 100-
µl reaction mixture containing 25 mM Tris acetate, pH 7.5, 3 mM magnesium
acetate, 2 mM ATP, 1 mM dithiothreitol and 100 µg/ml BSA, for 5 min at 30 °C.
RAD54 (20 nM) was added to the reaction mixture followed by a 1 min incubation.
The annealing was started with the addition of the 32P- labeled DNA strand (5 nM,
molecules). Aliquots (10 µl) were withdrawn at indicated time points and the
reaction was quenched by the addition of 5 µl stop solution (1.36% SDS, 1.4 mg/ml
proteinase K, 6% glycerol, 0.015% bromophenol blue) for 15 min at room
temperature. DNA products were analyzed by electrophoresis in 10%
polyacrylamide gels (17:1) in 1 x TBE buffer (90 mM Tris borate pH 8.3, and 1 mM
EDTA) at 23 °C. The gels were dried on DE81 chromatography paper (Whatman)
and quantified using a Storm 840 PhosphorImager (GE Healthcare).
97
3.3 RESULTS
3.3.1 RAD54 promotes annealing of complementary ssDNA strands
We tested the annealing activity of RAD54 using two complementary
oligonucleotides (#64 and #65, 48-mers), in which one of the oligonucleotide was
32P-labeled at the 5’-end (Figure 3.1 A). The non-labeled oligonucleotide was
incubated in the reaction mixture for 5 min at 30 °C. Then, RAD54 was added to
the reaction mixture and allowed to incubate for 1 min at 30°C. The annealing was
initiated with the addition of the labeled oligonucleotide. Aliquots were withdrawn
at indicated time points and the DNA products were analyzed by electrophoresis
in polyacrylamide gels. We found that RAD54 promoted annealing of
complementary ssDNA strands efficiently (Figure 3.1 B).
A no. 64 * RAD54 + * no. 65
98
B
80
RAD54 60
40
20 Spontaneous
% Annealed product Annealed % 0 0 5 10 15 20 time(min)
Figure 3.1 RAD54 promotes ssDNA annealing. A) the scheme for ssDNA annealing experiments. The ssDNA annealing was carried out between two 48- mer oligonucleotides (no. 64/65). The asterisk indicates the 32P-label. B) The kinetics of annealing of ssDNA (5 nM, each oligo) promoted by RAD54 (20 nM). The DNA products were analyzed by electrophoresis in 10% polyacrylamide gels.
3.3.2 Effect of nucleotide cofactors on RAD54 promoted annealing
RAD54 is a motor protein that translocates on DNA in an ATP-dependent manner. Most of the activities of RAD54 are dependent on its ability to hydrolyze
ATP, such as translocation, chromatin remodeling, and branch migration (Mazin et al., 2010). We wanted to determine if ATP hydrolysis was necessary for RAD54 promoted annealing activity.
99
We tested annealing activity of RAD54 in presence of ATP and ATPγS. We found that the annealing activity was atleast 2-fold lower in presence of ATPγS compared to ATP (Figure 3.2). This could suggest either that ATP hydrolysis is required for annealing activity or that nucleotide cofactor binding induces different conformational changes in the RAD54 protein affecting its annealing activity.
80 RAD54-ATP 60
40 RAD54-ATPS
20 Spontaneous-ATP
% Annealed product Annealed % Spontaneous-ATP S 0 0 5 10 15 20 time(min)
Figure 3.2 Effect of nucleotide cofactor on RAD54 annealing activity. The kinetics of annealing of ssDNA (5 nM, each oligo) promoted by RAD54 (20 nM) in presence of ATP or ATPγS. The DNA products were analyzed by electrophoresis in 10% polyacrylamide gels.
We wanted to test the annealing activity in presence of ADP nucleotide as
well. However, in presence of ADP, an increase in the spontaneous annealing of
100 ssDNA strands was observed. Therefore, we could not assess the effect of ADP nucleotide on annealing activity of RAD54.
3.3.3 N-terminal domain is necessary but not sufficient for annealing activity of RAD54
Recently, a second DNA binding site was reported in the N-terminal domain of RAD54 (Wright and Heyer, 2014) which led us to the hypothesis that RAD54 could promote annealing of ssDNA strands. We wanted to test if this second DNA binding site was required for annealing activity. We carried out annealing assays using two RAD54 mutants, RAD5496-747 and RAD541-142. We found that RAD5496-
747 had significantly reduced annealing activity, whereas Rad541-142 did not promote ssDNA annealing at all (Figure 3.3). Taken together, these results suggest that both the DNA binding sites are required for annealing activity of
RAD54.
101
80 RAD54 60
40
RAD5496-747 20 RAD541-142
% Annealed product Annealed % Spontaneous 0 0 5 10 15 20 time(min)
Figure 3.3 N-terminal domain is necessary but not sufficient for annealing activity of RAD54. The kinetics of annealing of ssDNA (5 nM, each oligo) promoted by RAD54, RAD5496-747, or RAD541-142 (20 nM). The DNA products were analyzed by electrophoresis in 10% polyacrylamide gels.
3.3.4 DNA-binding by N-terminal domian, not oligomerization, is required for
ssDNA annealing promoted by RAD54
In Chapter 2, we demonstrated that the RAD54 N-terminal domain is
essential for branch migration along with the conserved ATPase/DNA binding
domain. Our results indicated that the N-terminal domain is important for coupling
the RAD54 ATPase with branch migration through two connected, but distinct
steps: binding to HJs and DNA-dependent RAD54 oligomerization.
102
We wanted to test if both or one of the activity of the RAD54 N-terminal domain was involved in annealing activity promoted by RAD54. For this, we carried out annealing reactions in the presence of RAD54RKRK33-36AAAA and RAD54S49E mutant proteins; these two mutations affect DNA-binding by N-terminal domain and DNA-dependent oligomerization, respectively. We found that RKRK4A mutation strongly reduced ssDNA annealing of RAD54, whereas, S49E had no significant effect on this activity (Figure 3.4). These data support our earlier conclusion that RKRK4A and S49E represent the separation of function mutations, in which RKRK4A disrupts the N-terminal domain DNA binding, whereas S49E mutation specifically inhibits RAD54 oligomerization without a significant direct effect on DNA binding.
103
80 RAD54
60 RAD54S49E
40 RAD54RKRK33-36AAAA % annealing 20
Spontaneous 0 0 5 10 15 20 time (min)
Figure 3.4 DNA binding by RAD54 N-terminal domain is necessary for annealing activity. The kinetics of annealing of ssDNA (5 nM, each oligo) promoted by RAD54, RAD54S49E, or RAD54RKRK33-36AAAA (20 nM). The DNA products were analyzed by electrophoresis in 10% polyacrylamide gels.
3.3.5 RAD54 promotes annealing of complementary ssDNA and ssRNA strands
Recently, a novel mechanism has been proposed where RNA transcript can be used for homologous recombination and double-strand break repair (Keskin et al., 2014). The abundance of RNA transcripts in a cell makes this mechanism all the more interesting.
104
We wanted to further characterize the newly-found annealing activity of
RAD54, by testing its ability to anneal ssDNA and ssRNA in vitro. We carried out annealing reactions using complementary ssDNA and ssRNA, under conditions optimized for annealing of two ssDNA strands. We found that RAD54 can anneal ssDNA and ssRNA to form DNA-RNA hybrid (Figure 3.5).
40 RAD54
30
20
10
% Branch Migration Branch % Spontaneous
0 0 5 10 15 20 time (min)
Figure 3.5 RAD54 promotes annealing of ssRNA and ssDNA. The kinetics of annealing of ssDNA (5 nM) and ssRNA (5 nM) promoted by RAD54 (20 nM). The DNA products were analyzed by electrophoresis in 10% polyacrylamide gels.
3.4 DISCUSSION
105
RAD54 is a motor protein that belongs to the SWI2/SNF2 family of helicase- like proteins and is conserved in all eukaryotes. The helicase-like proteins use
DNA-dependent ATP hydrolysis to translocate along the DNA generating positive supercoils in the DNA ahead and negative supercoils behind the protein, allowing
DNA strands to transiently “breathe”. RAD54 is a versatile protein that links the homologous recombination process together by performing functions throughout.
(Heyer et al., 2006; San Filippo et al., 2008; Symington and Heyer, 2006; Tan et al., 2003).
In this study, we found another activity for the RAD54 protein. RAD54 can carry out annealing between two ssDNA strands or between ssDNA and ssRNA efficiently. The second DNA binding site in the N-terminal region of RAD54 (Wright and Heyer, 2014) is essential but not sufficient to carry out annealing, indicating the requirement for both DNA binding sites for annealing activity. In chapter 2, we found that the N-terminal domain binds preferentially to branched DNA structures, however, it can still bind to linear ssDNA or dsDNA. The binding of the N-terminal domain to ssDNA is associated with the annealing activity of RAD54. The activity depends on the presence of two DNA binding sites in RAD54, the N-terminal domain site, and the ATPase core site. The RKRK4A mutation and 95 aa N- terminal truncation that disrupt the N-terminal domain DNA binding strongly inhibit ssDNA annealing; similarly, RAD541-142 lacking the core DNA binding site is unable
to support the annealing activity. In contrast, the S49E mutation that specifically
inhibits protein oligomerization shows no significant effect on ssDNA annealing.
106
These data provide additional support to two distinct functions of the N-terminal domain during branch migration: DNA binding and protein oligomerization.
Previously, it was shown that several branch migration proteins, including
BLM and WRN, have ssDNA annealing activity (Cheok et al., 2005; Machwe et al.,
2005; Muftuoglu et al., 2008). Since this activity in most of the studied proteins, including RAD54 (Goyal et al, 2017, in preparation) is inhibited by RPA it unlikely plays a role in the cell, where RPA is abundant. Instead, it may manifest the presence of a secondary DNA binding site needed for branch migration in some cases because this site is also associated with protein oligomerization domain.
Thus, the presence of the secondary ATPase independent DNA binding site coupled with protein oligomerization seems to be a general requirement for the branch migration proteins (Cheok et al., 2005; Wu et al., 2005).
We also found that RAD54 can promote annealing of ssRNA and ssDNA to form an RNA-DNA hybrid. There is increasing evidence that suggests the possibility of using RNA transcripts as a template for homologous recombination and DNA repair (Keskin et al., 2014). RAD52 has been shown to be important for
RNA-transcript mediated homologous recombination, as it can also anneal ssRNA and ssDNA to form RNA-DNA hybrid (Keskin et al., 2014). In light of these recent developments, the annealing activity of RAD54 cannot be overlooked. Therefore, further work is required to establish the exact role of annealing activity of the
RAD54 protein in homologous recombination.
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Identification of small-molecule inhibitors of RAD54
4.1 INTRODUCTION
The Homologous recombination pathway is responsible for the repair of
DNA double-strand breaks, the most harmful types of DNA lesions, faithful
chromosome segregation during meiosis, and telomerase-independent telomere maintenance (Jasin and Rothstein, 2013; Krejci et al., 2012; San Filippo et al.,
2008). Homologous recombination uses homologous DNA molecules as a template to repair double-strand breaks and therefore is, generally, an error-free process.
RAD54, a key homologous recombination protein, is conserved among all eukaryotes. It is a multifunctional motor protein that is part of the SWI2/SNF2
ATPase family that not only stimulates strand exchange activity of RAD51 but also has its own independent functions along the homologous recombination pathway.
The SWI2/SNF2 family contains ATP-dependent chromatin remodeling proteins that can form multi-protein machines and their ATPase cores contain the seven conserved helicase domains that are associated with the Superfamily 2 helicases
(Alexeev et al., 2003; Hauk and Bowman, 2011; Singleton et al., 2007). RAD54 is believed to function throughout the process of homologous recombination. During pre-synapsis, RAD54 stabilizes the RAD51-ssDNA filament (Mazin et al., 2003;
Wolner and Peterson, 2005). In synapsis, RAD54 stimulates RAD51-mediated strand exchange and as an SWI2/SNF2 protein it can facilitate nucleosome sliding and induce changes in the DNA topology during the search for homologous
108 chromosomes (Alexeev et al., 2003; Alexiadis and Kadonaga, 2002; Jaskelioff et al., 2003). In post-synapsis, RAD54 can promote branch migration that serves to control the length of DNA exchanged between homologous DNA or dissociate
Holliday junctions (Bugreev et al., 2007; Bugreev et al., 2006b). RAD54 interacts with Mus81-Eme1 (Mms4), a structure-specific endonuclease, stimulating its DNA cleavage activity (Interthal and Heyer, 2000; Matulova et al., 2009; Mazina and
Mazin, 2008). RAD54 can also disrupt RAD51-dsDNA filaments which could be a final stage of the homologous recombination pathway to clear the DNA of proteins after the repair is completed.
While the biochemical activities of RAD54 are well characterized, its specific cellular functions remain to be elucidated. In this study, we wanted to identify specific small-molecule inhibitors of human RAD54 protein using high throughput screening of chemical libraries. The specific inhibitors could be used to study the functions of RAD54 in human cells. Since double-strand break- and inter-strand crosslink- inducing agents are commonly used in anticancer therapy, the specific
RAD54 inhibitors may also help to increase the therapy efficacy.
With the aim of better understanding the activities of RAD54 in vitro and to test their function in vivo, we used a FRET-based assay for high-throughput screening of a 400, 000 compound library in collaboration with The Broad Institute,
Cambridge, MA. The in vitro FRET-based primary assay was designed in a way that would result in increase in fluorescence as a result of DNA branch migration catalyzed by RAD54. In collaboration with the Broad Institute Probe Development
Center (BIPDeC), the assay was validated (Z’ >0.8) and used for identification of
109 candidate inhibitors of RAD54. We confirmed 55 small-molecule compounds that inhibited RAD54 branch migration activity using the optimized assay in our lab. Out of these 55 compounds, we identified two compounds, C-G01 and C-A23, which strongly inhibited branch migration activity without significantly affecting its
ATPase activity, similar to our N-terminal truncated RAD54 protein. Another compound C-E13 showed higher specificity for DT-40 chicken cells that were
RAD18 deficient. Previously, it has been shown that RAD18 and RAD54 knockouts are synthetically lethal in DT-40 chicken cells (Yamashita et al., 2002). Therefore,
C-E13 was used to generate the second-generation inhibitors to increase its potency.
4.2 MATERIALS AND METHODS
Chemicals, Proteins, and DNA
All oligonucleotides used in this study (Table 4.1) were purchased from IDT
Inc. and purified using denaturing 6-10% PAGE. To prepare oligonucleotide
dsDNA substrates, complementary ssDNA oligonucleotides were annealed as
described (Rossi et al., 2010) and stored at -20 °C. Oligonucleotides were labeled
using [γ-32P] ATP and T4 polynucleotide kinase. T4 polynucleotide kinase and restriction endonucleases were purchased from New England Biolabs.
PreScission™ Protease was purchased from GE Healthcare Life Sciences. The
small-molecule compounds were a gift from the Broad Institute Probe
Development Center.
110
Table 4.1. Sequences of the oligonucleotides used in this study *
Length in Number nucleotide Sequence, 5’3’ s
CTT TAG CTG CAT ATT TAC AAC ATG TTG ACC TAC AGC #71 94 ACC AGA TTC AGC AAT TAA GCT CTA AGC CAT CCG CAA AAA TGA CCT CTT ATC AAA AGG A
TCC TTT TGA TAA GAG GTC ATT TTT GCG GAT GGC TTA #169 93 GAG CTT AAT TGC TGA ATC TGG TGC TGT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT
T CCT TTT GAT AAG AGG TCA TTT TTG CGG ATG GCT TAG #170 94 AGC TTA ATT GCT AAA TCT GGT GCT GTA GGT CAA CAT GTT GTA AAT ATG CAG CTA AAG
#171 63 ACA GCA CCA GAT TTA GCA ATT AAG CTC TAA GCC ATC CGC AAA AAT GAC CTC TTA TCA AAA GGA
*Bold red letters indicate the nucleotides that form mismatched pairs in the branch migration products.
Purification of RAD54 from insect cells
Human RAD54 was purified from Sf21 cells using baculovirus expression
vector as described previously (Mazina and Mazin, 2004) with alterations indicated
below. The purification scheme included a Glutathione S-transferase (GST) affinity
column, followed by a Superdex 200 column, and then a Resource S column. In
this work, we used RAD54 constructs containing an amino-terminal GST affinity
tag separated from the RAD54 gene by a PreScission Protease™ recognition
111 sequence (L-E-V-L-F-Q-G-P). Following fractionation on the Superdex 200 column, the pool containing RAD54 was incubated with PreScission Protease (GE
Healthcare) (10 U of PreScission Protease per 100 mg of RAD54) for 4 h at 4 °C.
The sample was diluted to 100 mM KCl and loaded on a 1 ml Resource S column.
The fractions containing RAD54 or RAD54 mutants were analyzed for nuclease contamination, pooled, and stored in small aliquots at -80 °C. The protein appeared nearly homogeneous in a Coomassie-stained SDS-polyacrylamide gel.
ATPase Assay using Thin Layer Chromatography (TLC)
The phosphorylated or non-phosphorylated RAD54 protein (60 nM) was incubated with PX-junction (10 nM, molecules) and 8 mM ATP, 20 nCi of [γ-
32P]ATP in 10 µl of reaction buffer containing 25 mM Tris acetate, pH 7.5, 2 mM
DTT, 100 μg/ml BSA, 11 mM magnesium acetate at 30 °C. The level of ATP
hydrolysis was determined using TLC on PEI-cellulose plates in running buffer
containing 1 M formic acid and 0.3 M LiCl. The products of ATP hydrolysis were
quantified using a Storm 840 PhosphorImager (GE Healthcare).
112
Branch migration assay
PX junctions were prepared by annealing 32P-labeled forked DNA
intermediates (#71/169*) with 3’-tailed DNAs (#170/171) (50% molar excess of
cold tailed DNA) (Rossi et al., 2010). The PX sequence was designed so that one
mismatched bp would be generated during branch migration reducing the
spontaneous reaction. The RAD54 protein (60 nM, unless indicated otherwise)
was incubated with 32P-labeled synthetic PX-junction (32 nM, molecules unless
indicated otherwise), in a 100-µl branch migration buffer containing 25 mM Tris
acetate, pH 7.5, 3 mM magnesium acetate, 2 mM ATP, 1 mM dithiothreitol, 100
µg/ml BSA, the ATP-regenerating system (30 units/ml creatine phosphokinase and
10 mM creatine phosphate). The reactions were carried out at 30 °C. Aliquots (10
µl) were withdrawn at indicated time points, and DNA substrates were
deproteinized by treatment with stop solution (1.36% SDS, 1.4 mg/ml proteinase
K, 6% glycerol, 0.015% bromophenol blue) for 15 min at 37 °C. Samples were analyzed by electrophoresis in 8% polyacrylamide gels (29:1) in 1x TBE buffer (90 mM Tris borate, pH 8.3, and 1 mM EDTA) at room temperature. The gels were
dried on DE81 chromatography paper (Whatman) and quantified using a Storm
840 PhosphorImager (GE Healthcare).
CellTiter-Glo luminescent viability assay.
Wild type or RAD18-deficient DT40 cells were grown in log phase, following
which 3000 cells were seeded in 100 μl complete DMEM/F12 media containing
10% FBS, 1.5% chicken serum, 100U/ml penicillin, 100 μg/ml streptomycin and 10
113
μM β-mercaptoethanol in a white 96-well plate (Greiner Bio-One, cat# 655083).
Cells were allowed to grow overnight in a 37 ˚C, 5% CO2 incubator and then treated
with compounds in indicated concentrations. After 48h the cells were added with
30 μl/well of CellTiter–Glo reagent (Promega, cat# G7572) and incubated for 10
min at room temperature. The luminescent signal was read by GloMax®-Multi
Microplate Multimode Reader.
4.3 RESULTS
4.3.1 High-throughput screening for RAD54 inhibitors
We conducted a high-throughput screening of a 500, 000 compound library
in collaboration with The Broad Institute, MA to identify molecules that branch
migration activity of RAD54, using a fluorescence-based assay (Figure 4.1). The
assay utilizes a fluorescein molecule and a black hole quencher on the two strands
of a duplex DNA. If RAD54 is able to branch migrate the DNA substrate, the black
home quencher would be separated from the fluorescein molecule, and we would
observe fluorescence signal. However, if the small molecule is an inhibitor of
RAD54 branch migration activity, we would not observe any signal. This initial
screening identified 55 potential inhibitors of RAD54 branch migration activity.
114
Figure 4.1 Fluorescence-based assay for identification of RAD54 small- molecule inhibitors. Fluorescence-quenching assay for RAD54 branch migration. Experimental scheme. FLU-fluorescein; BHQ –black hole quencher.
4.3.2 Validation of hits identified by high-throughput screening
The hits identified by the high-throughput screening were then validated using the branch migration assay optimized in our lab. The hits were then grouped in terms of potency to inhibit the branch migration reaction. We found five compounds that showed >75% branch migration inhibition (shown in red), nine compounds that showed >50% branch migration inhibition (shown in blue), and nine compounds that showed >30% branch migration inhibition (shown in green)
(Figure 4.2). These compounds were also tested for their ability to inhibit ATP hydrolysis by RAD54 using the TLC-based method and for specificity using
RAD51-promoted strand exchange reaction as read-out (Table 4.2).
We found two compounds, C-G01 and A-23, which selectively inhibited branch migration activity of RAD54 and did not affect RAD54’s ATPase or RAD51’s strand exchange activity. The deletion of the N-terminal 95 aa of RAD54 did not
115 affect its ability to hydrolyze ATP, however, branch migration activity was almost reduced to zero. Because we observe similar effect with compounds, C-G01 and
C-A23, we hypothesize that these compounds function by interacting with the N- terminal domain of RAD54.
90 90
85 85
80 80
75 75
70 70
65 65
60 60
55 55
50 50
45 45
40 40
35 35 Branch migration (% ) 30 30
25 25
20 20
15 15
10 10
5 5
0 0 C08 C-I01 C-I03 C-I05 C-I07 C-I09 C-I11 C-I13 C-E01 C-E03 C-E05 C-E07 C-E09 C-E11 C-E13 C-E15 C-E17 C-E19 C-E21 C-E23 C-A01 C-A03 C-A05 C-A07 C-A09 C-A11 C-A13 C-A15 C-A17 C-A19 C-A21 C-A23 C-C01 C-C03 C-C05 C-C07 C-C09 C-C11 C-C13 C-C15 C-C17 C-C19 C-C21 C-C23 C-G01 C-G03 C-G05 C-G07 C-G09 C-G11 C-G13 C-G15 C-G17 C-G19 C-G21 C-G23 2% DMSO Compound Tested
Figure 4.2 Validation of 55 RAD54 inhibitors using branch migration assay. The effect of compounds (30 μM) on RAD54 (50 nM) branch migration of PX junctions (10 nM, molecules) over a 30 min reaction period. Red shows the five compounds that show >75 % branch migration inhibition. Blue shows the nine compounds that show >50 % branch migration inhibition. Green shows the 9 compounds in comparison with SN (shown in solid green) that show >30 % branch migration inhibition.
116
Table 4.2 IC50 values for compounds in specificity assays.
IC50 ratio Inhibitors, Broad ID BM, IC50, μMATPase, IC50, μM IC50 ratio (SE/BM) (ATPase/BM)
BRD-K70804394-001-10-8 0.37 6.8 18.4 81.1 C-G01
C--A23 BRD-K62090360-001-10-4 0.83 >12 >14.5 >602
C-E13 BRD-K84577567-001-09-7 2.7 3.6 1.3 21.1
C-A17 BRD-K27541991-001-02-4 4.7 34.7 7.4 >42.6
C-A13 BRD-K56525905-001-13-3 5.2 31.1 6.0 >38.5
C-E23 BRD-K94926671-001-02-2 6.6 6.6
C-C11 BRD-K08408435-001-10-6 7.2 8.1 1.1 16.1
C-E07 BRD-A04445933-001-11-9 8.4 >100 >11.9 >29.8
C-A15 BRD-K00370289-003-02-6 9.3 22.5 2.4 >21.5
C-A05 BRD-K74453742-001-11-7 9.8 34.1 3.5 48.9
C-I05 BRD-K72075906-003-02-7 10.1 53.4 5.3 10.6
C-G09 BRD-K87744949-001-03-2 12 6.9
C-G23 BRD-K33212518-001-07-7 13.7 >200 >14.6 14.6
C-A21 BRD-K36595427-001-11-4 16 68 4.3 >12.6
C-A19 BRD-K88534205-300-02-3 16.3 >200 >12.3 >12.3
C-C03 BRD-K64037573-001-09-8 20.1 >200 >10.0 >10
C-I01 BRD-K38997908-001-09-2 20.5 >200 >10 >9.8
C-E11 BRD-K01531637-001-12-4 22.9 220 9.6 11.6
C-C05 BRD-K02487325-001-10-9 23.7 >200 >8.4 >8.4
C-C07 BRD-K99032625-300-02-7 25.2 186 7.4 9.6
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4.3.3 Analysis of compounds in DT-40 chicken cells
Homologous recombination and Translesion DNA synthesis are two major pathways accounting for survival post-replication DNA damage. Translesion DNA synthesis fills the gap in the daughter strand after replication, whereas, homologous recombination utilizes intact sister chromatid as a template to repair gaps and breaks on the daughter strand. It was shown in DT-40 chicken cells, that
RAD18 and RAD54 genes are synthetically lethal (Yamashita et al., 2002). RAD18 is an essential protein for repair by Translesion DNA synthesis and RAD54 is involved in homologous recombination pathway. Therefore, we used RAD18 knockout chicken DT-40 cell line, to test the effect of the RAD54 inhibitors in cells.
We found four compounds, C-G01, C-A23, C-E13, and C-E07 that showed an atleast 2-fold reduction in the viability of RAD18 knockout cells compared to the wildtype DT-40 cells. One of these compounds, C-E13, showed more than 10-fold difference between viability of RAD18 knockout and wildtype cells. Therefore, we used compound C-E13 to generate the second generation of inhibitors for RAD54.
118
C-G01 C-E13 120 120 100 WT WT 5.8 M 100 RAD18 80 RAD18 2.5 M 80 60 60
40 Viability, % M 40 M Viability, % 20 20 4.80 0.45 0 0 0 10 20 30 40 50 0 5 10 15 Inhibitors, M Inhibitors, M
C-E07 C-A23
120 120 WT 6.9 M 100 100 RAD18 2.8 M WT 2.5M 80 80 RAD18 1.2M 60 60 40
Viability, % 40 Viability, % 20 20 0 0 0 5 10 15 20 25 0 5 10 15 Inhibitors, M Inhibitors, M
Figure 4.3 Effect of RAD54 inhibitors on the survival of RAD18-deficient and wildtype DT-40 cells. Wildtype and Rad18-deficient DT-40 cells were subjected to increasing concentrations of RAD54 inhibitors. The cell survivability was tested using CellTiter-Glo luminescent assay. The IC50 values for each inhibitor molecule are mentioned in the inset.
119
N
N O NH N N N N N
C‐G01 C‐E13
O O
N N N N N HN O NH S N
N N Cl
C‐E07 C‐A23
Figure 4.4 Structures of first generation RAD54 inhibitors
4.3.4 Screening of the second generation of inhibitors of RAD54
Using the compound, C-E13, as the base molecule (Figure 4.4), chemical modifications were introduced in its structure with an effort to increase its potency, oral bioavailability, solubility, chemical and metabolic stability, and minimal toxic effects.
We tested the 32 second generation inhibitors in the optimized branch migration assay. We found eleven compounds that showed >70% branch
120 migration inhibition (shown in red) (Figure 4.6), nine compounds that showed
>50% branch migration inhibition (shown in blue), and four compounds that showed >30% branch migration inhibition (shown in green). Moreover, we also found two compounds, A3 and B1, which stimulated the branch migration activity of RAD54 (Figure 4.5).
200
150
100
% Branch Migration% Branch 50
0 E1 E2 E3 E4 A1 A2 A3 A4 A5 A6 A7 B1 B2 B3 B4 B5 B6 B7 C1 C2 C3 C4 C5 C6 C7 D1 D2 D3 D4 D5 D6 D7 E13 2% DMSO Compounds Tested
Figure 4.5 Screening for 32 second generation inhibitors of RAD54. The effect of compounds (30 μM) on RAD54 (50 nM) branch migration of PX junctions (10 nM, molecules) over a 30 min reaction period. Red shows the eleven compounds that show >70 % branch migration inhibition. Blue shows the nine compounds that show >50 % branch migration inhibition. Green shows the four compounds that show >30 % branch migration inhibition.
121
C‐B3 C‐B6 C‐C2
C‐C3 C‐C5
C‐C6 C‐D7
C‐E1 C‐E4
Figure 4.6 Structures of second generation RAD54 inhibitors
122
4.4 DISCUSSION
RAD54, a key homologous recombination motor protein, is conserved
among all eukaryotes. Mutations in RAD54 have been linked to breast cancer,
colon cancer, and lymphoma (Matsuda et al., 1999). RAD54 is a multifunctional
protein that is believed to function throughout the process of homologous
recombination. This versatile role of RAD54 makes it a good target to regulate the
process of homologous recombination using small-molecule modulators. Over the
past few years, the use of small-molecule compounds has increased dramatically
because; (i) of the ease with which they could be applied and removed over
controlled period of time, (ii) they could target one specific function of the protein
rather than inhibiting the protein altogether, (ii) they help understand protein
functions in cell, and (iv) they could be used as drugs to treat specific diseased
conditions.
RAD54 functions throughout the process of homologous recombination,
however, what regulates it functions or interaction with other partner proteins is not
yet completely understood. Therefore, to better understand the regulation of
RAD54 activities, we used the “chemical genetics” approach, wherein, a small-
molecule modulator is used to regulate protein functions.
A high-throughput screening of a 400, 000 compound library was conducted
in collaboration with The Broad Institute, MA. The high-throughput screening assay
was designed to identify compounds specifically inhibiting branch migration activity
of RAD54. Branch migration is the process in which the Holliday junction
123 translocates along the DNA axis, progressively exchanging one strand of duplex
DNA for the homologous strand of another DNA duplex. It may either cause dissociation or extension of joint molecules, depending on the specific homologous recombination mechanism. It may also promote a restart of DNA replication, stalled at a DNA damage site by switching DNA template strands through a reversible regression of replication forks into Holliday junctions.
Branch migration is a complex process that, at minimum requires ATP- dependent DNA translocation and binding to Holliday junction. It is believed that most of the activities of RAD54 are dependent on its ability to hydrolyze ATP. We wanted to determine the mechanism of action of the 55 small-molecule inhibitors of RAD54 branch migration identified in the high-throughput screening. For this, we first wanted to confirm the small-molecule inhibitors using the optimized branch migration reaction in our lab, followed by testing their ability to inhibit ATP hydrolysis by RAD54.
We found that most of the compounds inhibited ATP hydrolysis of RAD54; however, we identified two compounds, C-G01 and C-A23, which inhibited branch migration without affecting ATPase activity. Furthermore, when tested for their ability to inhibit RAD54 in chicken DT-40 cells, C-G01 and C-A23, showed more than 2-fold difference in the survival assays for the RAD18-deficient cell line compared to wildtype cells. RAD18 and RAD54 knockouts have been shown to be synthetically lethal in DT-40 chicken cells (Yamashita et al., 2002). Another compound, C-E13, had IC50 10-fold lower for RAD18-deficient cells compared to
wildtype cells.
124
Therefore, compound C-E13 was used to develop second-generation of
RAD54 inhibitors by modifying its structure to improve its drug-like properties. A number of second-generation of inhibitors, when tested in the branch migration reaction, showed an increase in the potency for inhibiting RAD54 in vitro. These compounds need to be tested in DT-40 cells to establish their specificity.
Compounds, C-G01 and C-A23, did not affect RAD54’s ATPase activity but were selective in inhibiting its branch migration activity. We observed similar results with N-terminal truncated RAD54 protein (Chapter 2). We have previously shown that the N-terminal domain of RAD54 is essential for the branch migration activity of RAD54. RAD5496-747 was deficient in branch migration activity but had
2-3 fold higher ATPase activity. Moreover, we identified residues in the N-terminal
region that affected the DNA binding by N-terminal domain or the DNA-dependent
oligomerization by the N-terminal domain. Therefore, we hypothesize that these
two compounds, C-G01 and C-A23, interact with the N-terminal domain to inhibit
branch migration activity of RAD54. Further work is needed to test this hypothesis.
More importantly, we show that it is possible to inhibit one activity of RAD54 without
affecting other activities that are dependent on ATPase activity of RAD54. This
would enable to selectively inhibit one function of RAD54 and test its biological
function in cells.
125
CONCLUSIONS & FUTURE DIRECTIONS
Homologous recombination pathway is responsible for the repair of DNA double-strand breaks, the most harmful types of DNA lesions, faithful chromosome segregation during meiosis, and telomerase-independent telomere maintenance
(Krejci et al., 2012; San Filippo et al., 2008); (Jasin and Rothstein, 2013). It is critical for maintaining genome stability in all living organisms. Together with nucleotide excision repair, base excision repair, and non-homologous end joining, homologous recombination forms the house-keeping networks ensuring the stability of DNA.
Dysfunction of homologous recombination pathway causes genome instability that could lead to cancer and various chromosomal abnormalities such as Down’s and other syndromes causing premature aging diseases. Most of these disorders occur due to mutations in proteins such as the RecQ family helicases
BLM, WRN, and RECQ4 (Mohaghegh and Hickson, 2001); and the Fanconi’s anemia-related helicases FANCJ (Hiom, 2010) and FANCM (Whitby, 2010). The mutations in BRCA1 and BRCA2 proteins, involved in homologous recombination, result in genomic instability and predisposition to breast and ovarian cancer (Lok and Powell, 2012; Moynahan and Jasin, 2010). Therefore, making the studies on these proteins essential.
On one hand, we have gained quite a bit of knowledge on the initial steps of homologous recombination (Krogh and Symington, 2004; Li and Heyer, 2008) that includes: recognition of double-strand break site, resection of DNA with the
126 help of enzymes at the double-strand break site to generate DNA duplex with protruding 3’ single-stranded DNA tails. Followed by the formation of nucleoprotein filament between RAD51 and ssDNA, which searches for homologous DNA template, and strand invasion into the homologous duplex DNA forming the displacement loop or D-loop (Kowalczykowski, 2000; Sung et al., 2003). However, following joint molecule formation, the pathways that regulate the processing and resolution of these DNA intermediates are not well understood.
The choice of pathways during homologous recombination may revolve around the proteins that promote branch migration. Branch migration is the process in which one DNA strand is progressively exchanged for another strand of the homologous DNA. It may extend or shorten the heteroduplex DNA formed after DNA strand invasion, affecting the length of conversion tracks and thereby the amount of genetic information transferred between the two homologous DNA molecules (Pâques and Haber, 1999). Branch migration may cause dissociation of recombination intermediates, thereby, affecting the choice between a crossover and non-crossover pathway for homologous recombination to proceed (Bugreev et al., 2007). Branch migration may also a play role in restarting stalled replication forks during cell recovery after DNA damage (McGlynn et al., 2001; Michel et al.,
2004; Postow et al., 2001; Rothstein et al., 2000; Seigneur et al., 1998). RAD54, an important protein of the core homologous recombination machinery, can promote branch migration of Holliday junctions. Despite extensive studies, the mechanism of RAD54 branch migration activity is not well understood.
127
Therefore, the focus of my research was to understand the mechanism of
RAD54 promoted branch migration activity and how this activity is regulated in cells. Specifically, we wanted to determine the RAD54 domain that is responsible for its DNA-dependent oligomerization and the role of oligomerization in regulating
RAD54 activities.
Here, we demonstrate that the N-terminal domain of RAD54 is essential for its branch migration activity along with its “core” ATPase domain. In chapter 2, we showed that RAD54 mutant lacking a significant portion of the N-terminal domain,
RAD5496-747, is almost completely devoid of the branch migration activity.
However, this mutant still retains its ability to hydrolyze ATP and the ability to
translocate on DNA. We also found that the second DNA binding site in the N-
terminal domain promotes specific binding to Holliday junction substrates. RAD541-
142 shows a preference for Holliday junction-like DNA substrates similar to full
length RAD54. On replacing basic amino acid residues 33-36 by alanines
(RKRK33-36AAAA), we observed diminished DNA binding by the RAD54 N-
terminal domain suggesting the tentative position of the N-terminal domain DNA binding site. Interestingly, RAD5496-747 still retains the ability to recognize Holliday
junction-like structures, although the specificity for different branch DNA molecules
was changed. Together these results suggest the N-terminal domain area between
96 and 142 amino acid residues may also contribute to the recognition of Holliday
junction-like structures. The N-terminal domain of RAD54 may represent a
functional analog of the N-terminal domain of RecG or of the RecQ helicases
carboxyl terminal domain (RQC) that are responsible for specific binding to
128 branched DNA structures (Kim et al., 2013; Kitano et al., 2010; Pike et al., 2009 ;
Singleton et al., 2001). Furthermore, deletion the RAD54 N-terminal domain, similar to deletion of the BLM HRDC domain adjacent to RQC (Swan et al., 2014), increased the ATPase activity of the enzyme. It is thought that HRDC may suppress the BLM ATPase activity through direct interaction with the ATPase core.
For future studies, we would be interested in identifying the residues involved in recognition and binding to the Holliday junction-like substrates.
The proteolytic cleavage analysis of S. cerevisiae Rad54 shows that the N- terminal domain is unstructured in solution (Raschle et al., 2004). These data are consistent with the structural flexibility of the RAD54 N-terminal domain, which enables its interaction with various partners, like RAD51, CDK2, or DNA (Golub et al., 1997; Kettenbach et al., 2011; Raschle et al., 2004; Wright and Heyer, 2014).
In chapter 2, we further emphasized on the structural flexibility of the N-terminal domain of RAD54 by demonstrating a coupling between its binding to DNA and oligomerization. Previously, it was demonstrated that during branch migration
RAD54 forms oligomeric structures on Holliday junction substrates (Mazina et al.,
2007), a property shared with other branch migration proteins including BLM and
RuvAB (Mazina et al., 2012; West, 1997). However, the RAD54 domain responsible for oligomerization was unknown. We showed that the N-terminal domain of RAD54 is responsible for DNA-dependent oligomerization of RAD54.
The N-terminal domain alone is both necessary and sufficient for protein oligomerization and that the N-terminal truncation, significantly reduced the formation of RAD5496-747 oligomers in the presence of DNA. Moreover, we
129 identified mutations in the N-terminal domain that separately affect DNA binding and protein oligomerization. The RKRK33-36AAAA mutation decreased binding of
the RAD541-142 to DNA significantly. However, even this residual DNA binding
appeared to be sufficient to induce protein oligomerization observed in cross-
linking experiments with BMH. In contrast, S49E mutation only weakly inhibits
initial binding of RAD541-142 to DNA but disrupts the formation of large oligomeric
protein complexes visualized by BMH cross-linking. In full-size RAD54, each of
these mutations inhibited branch migration without affecting the ATPase activity of
the protein. The combination of these two mutations led to a stronger inhibition of
branch migration activity.
Taken together, these data led us to suggest a two-step process of
formation of the active RAD54 branch migration complex. First, RAD54 is targeted
to the Holliday junction by its N-terminal domain. The binding of RAD54 N-terminal
domain may induce a structural transition of Holliday junction to open conformation
that is proficient in branch migration. Second, binding of the RAD54 N-terminal
domain to DNA enables RAD54 oligomerization necessary for branch migration
(Mazina et al., 2007).
A phosphoproteomics study identified that RAD54 was phosphorylated at
Ser-49 (Kettenbach et al., 2011). Two CDK2 consensus sites are located in the
RAD54 N-terminal domain, with putative phosphorylation sites at Thr-31 and Ser-
49. Consistent with this, in vitro CDK2 phosphorylation data identified, T31 and
S49 as major phosphorylation sites in RAD54, as T31E, S49E double mutations
significantly reduced CDK2 phosphorylation of RAD54. We found that S49E, but
130 not T31E phosphomimetic mutation, inhibited oligomerization of RAD54 N-terminal domain. When incorporated in the full-size RAD54, this mutation inhibited the
RAD54 branch migration activity consistent with the important role of RAD54 oligomerization in its branch migration activity. The effect of the S49E mutation is specific for branch migration, as it had no significant effect on stimulation of
RAD51-promoted DNA strand exchange by RAD54 indicating that RAD54 oligomerization is not required for this stimulation. Phosphorylated RAD54 (in vitro by CDK2) also inhibited its branch migration activity, consistent with the results observed for the phosphomimetic S49E mutation.
RAD54 branch migration activity can lead to dissociation of D-loops, the product of ssDNA invasion into homologous DNA-template during double-strand break repair (Bugreev et al., 2007). This step of double-strand break repair, however, needs to be precisely controlled, as premature disruption of D-loops prior to their extension by DNA polymerase may abort double-strand break repair.
CDK2-dependent inhibition of RAD54 branch migration may help to delay D-loop disruption until the polymerization step is completed. Recently, it was also shown that Nek1 phosphorylates RAD54 at S572 in late G2 phase to modulate its interaction with RAD51 (Spies et al., 2016). Thus, multiple mechanisms are in place to control different RAD54 activities in the cell.
For future studies, we would be interested in determining the exact role of branch migration activity of RAD54 in homologous recombination pathway. We have identified mutations in the N-terminal domain of RAD54 that specifically affect its branch migration activity without affecting its ATPase activity. These mutations
131 could be introduced in the cells and their effect determined using DR-GFP assay, which measures the homologous recombination events taking place after the deliberate introduction of a double-strand break.
In chapter 3 we reported annealing activity for the multifunctional RAD54 protein. RAD54 can carry out annealing of two complementary single-stranded
DNA molecules or between single-strand DNA and single-strand RNA efficiently.
We found that the second DNA binding site in the RAD54 N-terminal domain was necessary but not sufficient for its annealing activity, indicating the requirement for two binding sites for annealing activity. RAD541-142 shows preference for branched
DNA structures, however, it can still bind to linear single-stranded or double- stranded DNA. This binding to single-stranded DNA by N-terminal domain is associated with annealing activity of RAD54.
Previously we show that the RKRK33-36AAAA mutation and 95 amino acids N-terminal truncation disrupt the DNA binding ability of N-terminal domain of
RAD54 and its ability to branch migrate Holliday junctions. These two mutations also disrupted the annealing activity of RAD54; similarly, RAD541-142 lacking the
core DNA binding site is unable to support the annealing activity. In contrast, the
S49E mutation that specifically inhibits protein oligomerization shows no significant
effect on ssDNA annealing (chapter 3). These data provide additional support that
RKRK33-36AAAA and S49E mutations separately affect DNA binding and protein
oligomerization, respectively by the N-terminal domain of RAD54.
132
The annealing activity has been reported for other several other branch migration proteins, including, BLM and WRN proteins. However, the annealing activity for most of these proteins is inhibited by RPA. Therefore, it is unlikely that it plays an important role in the cell where RPA is in abundance. Instead, it may manifest the presence of a secondary DNA binding site necessary for branch migration as in some cases this site is also associated with protein oligomerization domain. Thus, the presence of the secondary ATPase independent DNA binding site coupled with protein oligomerization may be a general requirement for the branch migration proteins.
We also report that RAD54 can anneal single-stranded DNA and RNA to generate DNA-RNA hybrid. The possibility of using RNA transcripts as a template for recombination and double-strand break repair (Keskin et al., 2014; Storici et al., 2007) makes this finding intriguing. Our lab in collaboration with Storici lab was successful in establishing the role of RAD52 in RNA-transcript dependent recombination (Keskin et al., 2014). Next, to gain more insight into this newly discovered RNA-transcript dependent recombination, we would be interested in assessing the possible role of RAD54’s annealing activity in the same.
Previously, streptonigrin was identified as an inhibitor of ATPase activity of
RAD54 (Deakyne et al., 2013). Strepatonigrin inhibited RAD54’s branch migration activity but had minimal effect on stimulation of strand exchange activity of RAD51.
This indicated that ATPase activity of RAD54 was dispensable for the stimulation of RAD51’s activity. Thus, streptonigrin helped to shed light on the involvement of
ATPase activity of RAD54 in stimulation of RAD51’s strand exchange activity
133
(Deakyne et al., 2013). However, streptonigrin was found to be cytotoxic and could not be used in further studies. In Chapter 4, we conducted a high-throughput screening to identify inhibitors of RAD54 branch migration activity. The small- molecule inhibitors could be used to better understand the functions of RAD54 in vitro as well test its function in vivo. For this, we developed a fluorescence-based assay in our lab to screen a ~400, 000 small-molecule compounds library in collaboration with The Broad Institute, MA. We identified 55 compounds from the initial screen that were further characterized using assays optimized in our lab. We identified two compounds, C-G01 and C-A23, which strongly inhibited the branch migration activity of RAD54 but had no significant effect on its ATPase activity. We believe that these compounds exert their action by interacting with the N-terminal domain of RAD54 because we observed similar results with N-terminal truncated
RAD54 protein. Therefore, we would like to determine the mechanism of action for these two compounds and study their interaction with the N-terminal domain of
RAD54 using various truncated and mutants of RAD54 protein. Moreover, these compounds could be used to study the exact function of branch migration in cells as they have no effect on the ATPase activity. Compared to streptonigrin’s structure that contained an active aminoquinone moiety, C-G01 contained a quinoxaline group that is isomeric with the quinazoline group in C-A23. These groups may be the active component of the inhibitors but unlike streptonigrin, they do not contain ketone groups within conjugated cyclic ring systems that play a role in generating reactive oxygen species. Therefore, these inhibitors may prove to be more successful in understanding RAD54’s cellular activities.
134
Previously, it was shown that RAD54 and RAD18 knockouts are synthetically lethal in chicken DT-40 cells (Yamashita et al., 2002). We identified another compound, C-E13, that was 10-fold more potent in killing RAD18-deficient
DT-40 cells compared to wildtype DT-40 cells. Thus, compound C-E13 was used to develop C-E13 analogs by substituting various chemical groups at the ortho-, para-, and meta-positions to increase its potency and minimize potential off-target effects. A number of second-generation of inhibitors, when tested in the branch migration reaction, showed an increase in the potency for inhibiting RAD54 in vitro.
These compounds need to be further tested in DT-40 cells to establish their specificity in vivo.
Another protein that we studied in the lab was RAD52 protein, which is responsible for DNA single-strand annealing sub-pathway of homologous recombination that is RAD51-independent. RAD52 can also promote D-loop formation, similar to RAD51, suggesting that it may potentially substitute RAD51 in some homologous recombination events. More importantly, mutations in RAD52 are synthetically lethal with mutations in BRCA1, BRCA2, PALB2, or in five RAD51 paralogs. The concept of synthetic lethality provides a framework for discovering drugs that selectively kill cancer cells. In chapter 5 we exploited synthetic lethality between the RAD52 and BRCA1&2 genes. We proposed that targeting RAD52 with small molecule inhibitors would disrupt the RAD52-dependent sub-pathway of homologous recombination in BRCA1- and BRCA2-deficient cells leading to cell death. Therefore, RAD52 represented an attractive potential therapeutic target as no RAD52 mutations or inactivation has been documented in human tumors. Using
135 fluorescence-quenching based assay for high-throughput screening, we identified
14 small-molecule compounds that specifically inhibited RAD52 ssDNA annealing and DNA pairing activities with IC50 better than 10 µM. We found that one of the compounds, D-I03, which had the strongest inhibitory effect in human cells was also selective in inhibiting the growth of CML patient cells where the expression of
BRCA1 is constitutively reduced. Also, D-I03 specifically disrupted the formation of RAD52 foci induced by cisplatin without affecting RAD51 foci in cells. In accord with specific targeting of RAD52 in human cells, D-I03 inhibited the single-strand annealing in USO2 cells, without significantly affecting canonical homology- dependent recombination.
Previously, it was demonstrated that the N-terminal domain is responsible for single-strand DNA annealing and DNA pairing activities of RAD52. Therefore, we suggest that D-I03 may specifically target this domain. However, further work is required to determine its mechanism of action. Our data suggests that RAD52 inhibitors can potentially be used as prototypes for development of novel therapies against hereditary breast cancer and ovarian cancers with defective BRCA1 or
BRCA2 proteins. In addition to this, RAD52 inhibitors can also be used to study its biochemical activities and cellular functions.
Taken together, our results demonstrate that the N-terminal domain of
RAD54 is essential for its branch migration activity. The N-terminal domain promotes: (i) specific binding of RAD54 to Holliday junction-like substrates, and (ii)
DNA-dependent oligomerization, necessary for the formation of active RAD54 branch migration complex. In addition, RAD54 N-terminal domain contains two
136 putative sites that could be phosphorylated by CDK2 in the cell. Phosphorylation at Ser-49 by CDK2 inhibits DNA-dependent oligomerization of the N-terminal domain and therefore, can regulate branch migration activity of RAD54 during the process of homologous recombination. Moreover, we show that inhibitors of homologous recombination proteins, RAD54 and RAD52, could be used to study cellular functions of these proteins as well for developing novel cancer therapies in combination with current therapies.
I believe that my contributions to this field have deepened our understanding of the RAD54 branch migration activity and the mechanisms that regulate branch migration. This would further help in defining its role in homologous recombination.
137
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Appendix A: Targeting BRCA1- and BRCA2- deficient cells with small-
molecule inhibitors of RAD52
A.1 ABSTRACT
RAD52, an evolutionarily conserved protein from yeast to humans, is a member of Homologous recombination pathway and plays a role in genome maintenance. In contrast to yeast Rad52 which is crucial for most of the homologous recombination events, RAD52 knockout shows no significant phenotypic effect in mammals. However, recent work demonstrated that combination of RAD52 mutation with mutations in genes that cause hereditary breast cancer and ovarian cancer like BRCA1, BRCA2, PALB2, and RAD51C results in lethality. In this study, to use RAD52 as a potential therapeutic target for cancer therapy we screened a 372,903-compound library to identify small molecule inhibitors using a fluorescence-quenching assay for ssDNA annealing activity of RAD52. Further characterization using biochemical and cell-based assays of the obtained 70 alleged inhibitors led to the identification of compounds that specifically inhibit the biochemical activities of RAD52, selectively inhibit the growth of BRCA1- and BRCA2-deficient cells and inhibit RAD52-dependent single- strand annealing in human cells. In future, these identified compounds can be used for development of novel cancer therapy and as a probe to study mechanisms of
DNA repair.
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A.2 INTRODUCTION
DNA repair is crucial for maintaining genomic integrity in all organisms.
Multiple DNA repair systems evolved to eliminate a broad variety of DNA lesions
caused by different exogenous agents or genotoxic products of metabolism.
Homologous recombination is one such highly conserved DNA repair mechanism
pathway that is involved in repair of double-stranded breaks, the most harmful form
of DNA damage, which are induced by ionizing radiation and other agents or endogenously due to stalled replication forks, DNA interstrand crosslinks, and incomplete telomere synthesis. Even though there are other alternate pathways
like non-homologous end joining to repair double-strand breaks, they are not error-
free and precise like homologous recombination as it utilizes an intact homologous
DNA strand as a donor template for the repair. (Pâques and Haber, 1999; San
Filippo et al., 2008)
The process of homologous recombination involves recognition of a double-
strand break, enzymatic processing of the break site to produce 3’-ssDNA tails,
the formation of RAD51-ssDNA filament that searches for homology and promotes
strand invasion into the homologous duplex DNA leading to the formation of
displacement loop (D-loop) intermediate. After this step, homologous
recombination may either proceed via synthesis-dependent strand annealing or
double-stranded break repair mechanism. In synthesis-dependent strand
annealing, D-loops dissociate and the extended invading strand re-anneals with
163 the resected second end of double-strand break forming non-crossover recombinants, whereas in double-strand break repair, the displaced strand of the
D-loop may anneal with the second resected end of the double-strand break leading to formation of Holliday junctions, their branch migration, and resolution to form crossover recombinants. (Allers and Lichten, 2001; Pâques and Haber,
1999).
Due to its essential role in DNA repair mechanism, mutations in proteins involved in homologous recombination, most commonly BRCA1 and BRCA2, result in genomic instability and predispose to breast and ovarian cancer (Lok and
Powell, 2012; Moynahan and Jasin, 2010). In this case, when homologous recombination is defective the BRCA1- and BRCA2- deficient cancer cell viability depends on the remaining alternative DNA repair mechanisms (Helleday, 2011;
Lok and Powell, 2012; Lord and Ashworth, 2012). Thus, it was demonstrated that
PARP1, a protein involved in DNA damage signaling and repair of DNA single- stranded breaks, is essential for viability of cancer cells that are deficient in the homologous recombination pathway (Bryant et al., 2007; Farmer et al., 2005).
Furthermore, hereditary breast cancer and ovarian cancer cells, deficient in
BRCA1 or BRCA2 can be eliminated using PARP1 inhibitors with a minimal harm to normal cells with at least one copy of functional BRCA1/2 genes (Helleday,
2011; Lok and Powell, 2012; Lord and Ashworth, 2012).
New findings demonstrated that cells deficient in either BRCA1/2, PALB2
(partner and localizer of BRCA2) or five RAD51 paralogs, including RAD51C, when combined with a deficiency in recombination factor RAD52, are synthetic lethal
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(Chun et al., 2013; Feng et al., 2011; Lok et al., 2013). Mutations in PALB2 and
RAD51C also contribute to hereditary breast and ovarian cancer (Antoniou et al.,
2014; Meindl et al., 2010). Both PARP1 and RAD52 play different roles in maintaining cell viability in BRCA1/2-deficient and PALB2-deficient cells. PARP1 is involved in repair of DNA SSBs. During DNA replication, unrepaired SSBs may cause the formation of DNA double-stranded breaks or stalled replication forks which are repaired by the homologous recombination pathway. BRCA1/2/PALB2, constitute the major sub-pathway of homologous recombination; mutations in these proteins disrupt homologous recombination making hereditary breast and ovarian cancer cells vulnerable to PARP1 inhibitors. However, recent data indicate that in addition to the BRCA1/2/PALB2 sub-pathway, the secondary homologous recombination sub-pathway operates in a mammalian cell that depends on RAD52 protein (Feng et al., 2011; Lok et al., 2013). In normal mammalian cells, this pathway plays a minor role, as RAD52-/- mice are viable and fertile and do not display DNA damage sensitivity, abnormalities, or significant cancer predisposition
(Rijkers et al., 1998). However, this sub-pathway becomes essential for viability in cells that lack the BRCA1/2/PALB2 sub-pathway. Thus, these findings identify
RAD52 as a potential therapeutic target for familial breast cancer, familial ovarian cancer, and other types of cancer with defective BRCA1/2/PALB2 genes. Keeping this concept in mind, one of the co-authors (T.S.) demonstrated that inhibition of
RAD52 DNA binding activity by small peptide aptamer exerted synthetic lethality in BRCA1- and BRCA2-mutated cancer cells without any detectable side effects in normal cells and in mice (Sullivan et al., 2016).
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Here, in order to develop small molecule inhibitors of the RAD52 ssDNA annealing activity, we performed a high throughput screening of a 372,903- compound library using a fluorescence-quenching assay. We then characterized the obtained 70 alleged RAD52 inhibitors using biochemical and cell-based assays. We found several compounds that specifically inhibit the biochemical activities of RAD52 and growth of BRCA1- and BRCA2- deficient cells. One of these compounds, D-I03, also specifically inhibited RAD52-dependent single- strand annealing in human cells. We will use these compounds for the development of novel cancer therapy and also as a probe to study the mechanisms of DNA repair in human cells.
A.3 MATERIAL AND METHODS:
Chemicals, proteins, and DNA.
Cisplatin was purchased from Sigma-Aldrich. Human RAD52 and RAD51
(Figure A.1) were purified as described (Bugreev et al., 2005). The
oligonucleotides (Table A.1) were purchased from IDT, Inc and further purified by electrophoresis (Rossi et al., 2010). Supercoiled pUC19 pCBASce and pMX-GFP plasmid DNAs were purified using Qiagen kits. All DNA concentrations are expressed as moles of nucleotide.
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Figure A.1 Analysis of RAD51 and RAD52 proteins in an SDS-polyacrylamide gel. Proteins were stained with Coomassie blue. Lane M, Migration markers; Lane 1, Rad52; Lane 2, RAD51. 1μg of each protein was loaded on a 12% SDS- polyacrylamide gel.
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Table A.1 Sequences of the oligonucleotides used in Appendix A.
Length, N Sequence (5’→3’) nt FLU-CACTGTGATGCACGATGATCGACGACAGTAGTCAGT 337- 60 GCTGGGTCAACATCTGTATGCAGG FLU
AGCACTGACTACTGTCGTCGATCATCGTGCATCACAGTG– 1337- 39 BHQ1 BHQ1
ATACAGATGTTGACCCAGCACTGACTACTGTCGTCAATCAT 265-55 55 CGTGCATCACAGTG
CGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCA GATTGTACT GAG AGT GCA CCA TAT GCG GTG TGA AAT 90 90 ACC GCA CAG ATG CGT
Note: “FLU” and “BHQ1” denote Fluorescein and Black Hole Quencher 1, respectively
Compound libraries, compounds.
We used a 93672-compound Broad’s diversity-oriented synthesis (DOS)
library and a 279,231-compound Molecular Libraries Probe Center Network
(MPLCN) library. All the compounds were dissolved in DMSO (Sigma, Cat #
D8418). In the working solutions, the DMSO concentration added with the stock of
168 compounds was 2% (v/v) unless indicated otherwise. The compounds for confirmation analysis were purchased from Asinex Ltd., ChemBridge Co.,
ChemDiv Inc, Enamine, FCH Group, Frontier Scientific Services Inc.,
InterBioScreen Ltd., Life Chemicals Inc., Scientific Exchange Inc., Sigma-Aldrich
Co., and Vitas-M Laboratory Ltd.
The fluorescence-quenching assay for RAD52 DNA annealing.
Tailed dsDNA substrate was prepared by thermal annealing of ssDNA oligonucleotides 337-F and 1337-BHQ1 containing Fluorescein and Black Hole
Quencher 1 residues at the 5’ and 3’ end, respectively. DNA annealing was initiated by adding RAD52 (20 nM) to the mixture of ssDNA oligonucleotide 265-
55 (5 nM, molecules) and tailed dsDNA 337-F/1337-BHQ1 (5 nM, molecules) in buffer containing 25 mM Tris-acetate pH 7.5, 100 µg.ml-1 BSA and 1 mM DTT. The
fluorescence intensity was measured in a 3-mm quartz cuvette (Starna Cells) using a FluoroMax-3 (HORIBA) fluorimeter with 492 nm excitation wavelength and 520 nm emission wavelength at 30 °C for at least 2000 s.
High-Throughput Screening for RAD52 inhibitors.
The fluorescence-quenching assay for RAD52-promoted DNA annealing
was optimized to a 4 μl 1536 well protocol using 25 nM RAD52 and 8 nM
(molecules) DNA in buffer containing 25 mM Tris-acetate pH 7.5, 100 µg.ml-1 BSA,
1 mM DTT, and 0.01% Pluronic F-68. Wells containing no RAD52 were used as
169 a positive control to estimate the activity of fully inhibited protein; wells in which the compounds were replaced with only the vehicle (DMSO) were used as a neutral control. The HTS was performed using the 8 channel BioRAPTR 1536 (Beckman) for reagent dispensing. The reactions were carried out for 30 minutes followed by measurement of an endpoint fluorescence (485 nm excitation, 535 nm emission) using an EnVision multimode plate reader (Perkin Elmer). Wells containing no
RAD52 enzyme were used to as positive control, and data were analyzed using
Genedata. The compounds with an inhibitory effect of 30% or greater were tested further by measuring the concentration dependence (in a range from 1 nM to 100
μM) of their inhibition of RAD51. The most potent inhibitory compounds were analyzed further using non-fluorescent assays. Detailed methods for RAD52 screening are in PubMed: https://pubchem.ncbi.nlm.nih.gov/bioassay/651660#section=Top
The D-loop formation by RAD52 or RAD51.
To form RAD52 nucleoprotein complexes, RAD52 (0.45 μM) was incubated with a 32P-labeled ssDNA (oligo 90) (3 μM, nt) in buffer containing 25 mM Tris-
Acetate, pH 7.5, 100 μg.ml-1 BSA, 0.3 mM magnesium acetate, and 2 mM DTT at
37 °C for 15 min. To form RAD51 nucleoprotein filament, RAD51 (1 0.45 μM) was incubated with 32P-labeled ssDNA (1.35 μM, nt) in buffer containing 25 mM Tris-
Acetate, pH 7.5, 100 μg.ml-1 BSA, 1 2 mM calcium chloride, 1 mM ATP and 2 mM
DTT for 15 min at 37 °C. Then inhibitors were added to both reactions and incubation continued for 15 min at 37 °C. D-Loop formation was initiated by
170 addition of supercoiled pUC19 DNA (50 μM or 22.5 μM, nucleotides, for RAD52 and RAD51-promoted reactions, respectively) and was carried out 15 min at 37
°C. The reactions were stopped and deproteinized by the addition of 1.5% SDS and proteinase K (0.8 mg/ml) for 15 min at 37 °C, mixed with a 0.10 volume of loading buffer (70% glycerol, 0.1% bromophenol blue), and analyzed by electrophoresis in 1% agarose gels in TAE buffer (40 mM Tris acetate, pH 8.3, and
1 mM EDTA) at 5 V/cm for 3 h. The gels were dried on DEAE-81 paper (Whatman) and the yield of D-loops quantified using a Storm 840 PhosphorImager and
ImageQuant 5.2 (GE Healthcare). The D-loop yield was expressed as a percentage of plasmid DNA carrying D-loops relative to the total plasmid DNA.
To form RAD51 nucleoprotein filament, RAD51 (0.45 μM) was incubated with 32P-labeled ssDNA (1.35 μM, nt) in buffer containing 25 mM Tris-Acetate, pH
7.5, 100 μg/ml BSA, 1.5 mM calcium chloride, 1mM ATP and 2 mM DTT for 15 min at 37 °C. Then inhibitors were added to both reactions and incubation continued for 15 min at 37 °C. D-Loop formation was initiated by addition of supercoiled pUCFBR DNA (22.5 μM, nt) and was carried out 15 min at 37 °C.
Calculation of the IC50 value for RAD52 inhibitors.
IC50 values were calculated using GraphPad Prism V5.0 software. The data were obtained from three independent repeats of experiments.
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Acridine orange displacement assay for DNA binding.
To rule out DNA binding as an undesired mechanism of action, we tested the ability of the selected compounds to bind DNA. The compounds in varied concentrations were added into 30 μl reaction mixtures containing 50 nM acridine orange and 6 μg/ml salmon sperm DNA, 10 mM HEPES, pH 7.5; 1 mM EDTA pH
7.5; 100 mM NaCl in 384-well plates, and the reactions were incubated at room temperature for 20 min followed by fluorescence polarization measurement using an EnVision (Perkin Elmer) equipped with a 480 nm excitation filter and 535 nm S and P emission filters with a D505 FP/D535 dichroic mirror. Mitoxantrone (10 μM) was used as a positive control. The S and P values are processed with the standard fluorescence polarization calculation formula (mP=1000*(S-
G*P)/(S+G*P) where G is the G-factor and is approximately 1.
Luminescent cell viability assay.
BxPC3 cells were kept in RPMI 1640 (ATCC) media supplemented with
10% FBS (Gibco); Capan-1 cells were kept in IMDM (ATCC) media containing
20% FBS (GIBCO); UWB1.298 and UWB1.298 (BRCA1+) cells were kept in
48.5% RPMI1640 (ATCC), 48.5% MEGM (Clonetics/Lonza, MEGM kit, CC-3150) and 3% FBS (GIBCO) respectively. Cells in log-phase were harvested and 100 µl cell suspensions were re-plated in a 96-well plate with a final density of 4000 cells/well. After overnight growth, cells were treated with indicated concentrations of compounds. Media containing the invariant concentration of compounds were refreshed every 3 days until cells were finally lysed by 30 μl/well of Promega
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CellTiter-Glo reagents and read on a Promega GloMax 96 reader on day 10 (9 days exposure). Promega CellTiter-Glo protocol is available on the web: http://www.promega.com/resources/protocols/technical-bulletins/0/celltiter-glo- luminescent-cell-viability-assay-protocol/
The clonogenic survival assay.
MDA-MB-436 cells were cultured in RPMI + 10% FBS. BRCA-proficient and
BRCA-deficient cells were plated on day 0 in triplicate at 5,000 cells/well. On days
1 and 3, the cells were treated with 0, 2.5 μM, 5 μM, or 10 μM D-I03, D-G23 or D-
G09. Cells were counted on day 4 on a hemocytometer, using Trypan Blue exclusion, and immediately were plated in a clonogenic assay at a density of 500 cells/well in a 6 well plate, in RPMI + 10% FBS. After two weeks, the colonies were fixed/stained with 0.05% of 10 mg/ml ethidium bromide in 50% ethanol and visualized with Alphaimager gel imager (Alpha Innotech).
CML viability assay.
Lin-CD34+ primary CML and normal cells were obtained by magnetic sorting using the EasySep negative selection human progenitor cell enrichment cocktail followed by treatment with human CD34 positive selection cocktail
(StemCell Technologies), and were subsequently cultured in StemSpan H3000 media (StemCell Technologies) supplemented with a cocktail of growth factors
(100 ng/mL stem cell factor, 20 ng/mL interleukin3 [IL-3], 100 ng/mL fms-related tyrosine kinase 3 ligand, 20 ng/mL granulocyte colony-stimulating factor, 20 ng/mL
173
IL-6). For the viability assay, CD34+ CML (n=3) and normal (n=5) cells were plated at 1x104 cells/well in 96 well plates on day 0, and treated with 0 μM, 2 μM, 5 μM, or 10 μM D-I03 on days 0 and 2. Viable cells were counted on day 4 using Trypan
Blue staining.
Measurement of compound binding to RAD52 by SPR.
Experiments were performed using the ProteOn XPR36 SPR array system
(Bio-Rad). ProteOn GLH sensor chips were preconditioned with two short pulses
each (10 s) of 50 mM NaOH, 100 mM HCl, and 0.5% SDS. Then the system was
equilibrated with PBS-T buffer (20 mM Na-phosphate, 150 mM NaCl, and 0.1% polysorbate 20, pH 7.4). Individual ligand flow channels were activated for 5 min at 25 °C with a mixture of 1-ethyl-3-[3-dimethylaminopropyl carbodiimide hydrochloride) (0.2 M) and sulfo-N-hydroxysuccinimide (0.05 M). Immediately after chip activation, either RAD52 (100 µg.ml-1 in 25 mM Tris-Acetate, 20 mM KCl, 0.3
mM magnesium acetate, pH 7.5) or the anti-HIV mAb 2F5 (100 µg.ml-1 in 10 mM
sodium acetate, pH 5.0) was injected across ligand flow channels for 5 min at a
flow rate of 30 µl.min-1.
Excess active ester groups on the sensor surface were capped by a 5-min
injection of 1 M ethanolamine HCl (pH 8.5). This resulted in the coupling of RAD52
and 2F5 at a density of 9,000 RUs (response unit, which is an arbitrary unit that
corresponds to 1 pg/mm2). The standard deviation in the immobilization level from
the six spots within each channel was less than 4%. Compounds in indicated
concentrations in 25 mM Tris-Acetate, 20 mM KCl, 0.3 mM magnesium acetate,
174 pH 7.5, supplemented with 0.005% polysorbate 20 and 2% DMSO were injected over the control and RAD52 surfaces at a flow rate of 200 µl min-1, for either a 30s
(D-I09) or 1-min association phase (D-I03, D-G23), followed by a variable dissociation phase at 25 °C using the "one-shot" functionality of the ProteOn
(Bravman et al., 2006).
Specific regeneration of the surfaces between injections was not needed owing to the nature of the interaction. Data were analyzed using the ProteOn
Manager Software version 3.0 (Bio-Rad). The responses of a buffer injection and responses from the reference flow cell with the anti-HIV mAb 2F5 were subtracted to account for nonspecific binding. Experimental data were fitted globally to a simple 1:1 binding model. The average kinetic parameters (association [ka] and dissociation [kd] rates) generated from three data sets were used to define the equilibrium dissociation constant (KD). Data that could not be adequately fitted to a binding model were analyzed using equilibrium analysis, plotting the response at equilibrium versus concentration and fitting to a steady state model.
Measuring the effect of inhibitors on GFP-RAD52 and RAD51 foci formation.
GFP-RAD52 foci formation was measured in a BCR-ABL1-positive BRCA1- deficient 32Dcl3 murine hematopoietic cell line that expresses GFP-RAD52
(Cramer-Morales et al., 2013). RAD51 foci formation was measured in parental
32Dcl3. Both cell lines were. The cells were cultured in IMDM plus 10% FBS. The cells were plated at 500,000 cells/ml and pretreated for 4 h with either D-G23 or
D-I03 (2.5 μM) for GFP-RAD52 foci or with D-I03 (2.5 µM) for RAD51 foci (or no
175 pretreatment for the control and cisplatin-treated cells). After 4 h of incubation, the cells were treated with 3 μg/mL cisplatin for 16 h. Following cisplatin treatment, cytospins were prepared using polylysine coated slides (Thermo Scientific). DNA was counterstained with DAPI. To detect RAD51 foci, cells were stained with an anti-RAD51 antibody (Thermo Scientific), followed by a secondary antibody conjugated with AlexaFluor 594. RAD51 and GFP-RAD52 foci were visualized with an inverted Olympus IX70 fluorescence microscope equipped with a Cooke
Sensicam QE camera (The Cooke Co., Auburn Hills, MI, USA). Images from 25-
60 cells/group were processed using SlideBook 3.0 (Intelligent Imaging
Innovation).
Measuring the effect of D-I03 on single-strand annealing and gene conversion in U2OS cells.
U2OS cells with the chromosomally integrated single-strand annealing
(U2OS-SSA) or gene conversion HDR (U20S-HDR)-reporter were cultured in
DMEM (Sigma D-6429) containing 10% FBS (Gibco) supplemented with antibiotics (penicillin 100 U/ml, streptomycin 100 μg/ml, and plasmocin 2.5 μg/ml
(Gunn et al., 2011; Gunn and Stark, 2012). At 80% confluence, cells were trypsinized and plated in triplicate at a density of 2x105 cells/well in 6 well plates.
After 22 h cells, the wells were washed with 1X PBS and further incubated for 2 h in antibiotic-free DMEM-10% FBS. Cells were transfected with pCBASce (0.8 μg) expressing I-SceI endonuclease or, in controls, with pUC19 (0.8 μg) or pMX-GFP
(0.8 μg) plasmids using Lipofectamine 2000. After 3 h of transfection, cells were
176 washed with antibiotic-free DMEM-10% FBS. Then, cells were incubated in
DMEM-10% FBS supplemented with antibiotics and containing D-I03 at indicated concentrations followed by additional incubation for 48 h. In each well, cells were washed with 1X PBS, trypsinized, and fixed with 3.3 % formaldehyde. Fixed cells were kept on the ice. The yield of GFP+ positive cells was measured by flow cytometry using Guava EasyCyte PRO (EMD Millipore).
A.4 RESULTS
A.4.1 High-throughput screening for RAD52 inhibitors.
In order to target hereditary breast cancer and ovarian cancer cells, we wanted to develop small molecule inhibitors of RAD52 using high throughput screening (HTS) of compound libraries. In vitro, RAD52 carries out annealing of complementary ssDNA molecules and an invasion of ssDNA into homologous duplex DNA. To screen for inhibitors of RAD52 ssDNA annealing activity, we developed a fluorescence-quenching assay (Figure A.2). In this assay, RAD52 promotes DNA annealing between synthetic ssDNA (Oligo 265-55; 55 nt) and tailed dsDNA (tdsDNA) composed of a ssDNA 60-mer (Oligo 337-FLU) carrying fluorescein (FLU), a fluorescence donor group, and an ssDNA 39-mer (1337-
BHQ1) carrying black hole quencher 1 (BHQ1), a non-fluorescent acceptor group
(Figure A.2A). The RAD52-promoted reaction included two steps: 1) annealing between Oligo 265-55 and a 15-nt ssDNA region of the tdsDNA and 2) displacement of the ssDNA 1337-BHQ1 strand from the tdsDNA resulting in an increase in fluorescence due to secession of fluorescence quenching by the BHQ1
177 group (Figure A.2B; an example of the effect of D-I03 inhibitor on RAD52-promoted ssDNA annealing is shown in Figure A.2C and Figure A.3). Of these two steps, annealing was the limited step of the reaction in our assay, since RAD52 promotes
DNA strand exchange between ssDNA and blunt end dsDNA poorly (Bi et al.,
2004). In order to decrease RAD52-independent (uncatalyzed) reaction during branch migration step, we incorporated a single mismatch in Oligo 265-55 ssDNA
(Figure A.2A).
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Figure A.2 Identification and characterization of RAD52 small molecule inhibitors. (A) Fluorescence-quenching assay for RAD52 ssDNA annealing (plus DNA strand displacement). Experimental scheme. FLU-fluorescein; BHQ 1–black hole quencher 1. DNA substrates contain a mismatch (denoted by x) to block spontaneous reaction. (B) The kinetics of ssDNA annealing measured on a FluoroMax3 fluorimeter. The fluorescence intensity was expressed in arbitrary units (AU). Shown is the representative result; the reactions were repeated at least three times. (C) RAD52 annealing activity was measured in the presence of D-I03 in indicated concentrations. (D) The scheme of the D-loop assay: RAD52 forms a complex with ssDNA and promotes its homologous pairing with pUC19 plasmid
179
DNA. Asterisk denotes 32P label on ssDNA. (E) Inhibition of the RAD52 DNA pairing activity by tested compounds (30 µM). Error bars indicate standard deviation (SD).
Figure A.3 The effect of D-I03 concentration on the initial rate of ssDNA annealing promoted by RAD52. The rates of RAD52-promoted ssDNA annealing were calculated based on the data in Figure A.2C. The data are the mean of 3 independent measurements; error bars represent the SD.
The fluorescence-quenching assay was optimized for 1536 well plates (Z’
Avg = 0.64). The primary HTS of the 372,903-compound library yielded 1687 positive hits that caused more than 30% inhibition of the RAD52-dependent fluorescence increase (0.5 % activities), including 628 hits in a 93672-compound
Broad’s diversity-oriented synthesis (DOS) library (0.7% activities) and 1115 hits in a 279,231-compound Molecular Libraries Probe Center Network (MPLCN) library (0.4% Activities). The hits were further analyzed for a concentration dependence in inhibiting of RAD52 and by testing their DNA binding affinity using the acridine orange assay. As a result, 187 compounds were identified that
180
inhibited RAD52 with the IC50 lower than 10 μM and displayed no DNA binding.
These remaining compounds were assessed for their potential chemical tractability
by removing compounds with highly reactive or unstable functional groups and
focusing on chemotypes that were synthetically accessible and attractive. The
selected compounds, as well as some closely related new analogs of certain hits,
were next purchased as dry powders from commercial sources. After executing this selection process, 70 compounds were obtained for further analyses.
We tested the inhibitory effect of the 70 selected compounds using the D- loop assay, in which RAD52 promotes pairing between 32P-labeled ssDNA and
homologous supercoiled plasmid pUC19 DNA (Figure A.2D); the product of the
reaction, D-loops, were analyzed by electrophoresis in 1% agarose gels. First, by
testing the effect of the selected compounds at fixed (30 μM) concentration we identified 17 compounds that inhibited D-loop formation by more than 90% (Figure
A.2E). Then, we measured the effect of each of the 17 compounds on RAD52-
dpendent D-loop formation in a concentration dependent manner. The IC50 of
these compounds varied in a range between 2.7 μM and 17.5 μM (Figure A.4A-C;
Table A.2). In the D-loop assay, the IC50 values were generally higher than in the
fluorescence-quenching assay likely due to the higher RAD52 concentration
employed by the former assay, 450 nM vs 25 nM, and due to the different nature
of the assays.
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Figure A.4 The effect of inhibitors on DNA pairing activity of RAD52. (A) The products of DNA pairing (D-loops) were analyzed by electrophoresis in 1% agarose gels. (B) Graphical representation of the data from A. (C) The IC50 values for 17 tested compounds. (D) The effect of the RAD52 inhibitors on DNA pairing activity of RAD51. The effect was measured using the D-loop assay at the inhibitors concentrations that correspond to their 10 x IC50 values (27 μM - 176 μM) for RAD52 pairing activity; concentrations of A05 and B02 were 100 μM. Error bars indicate SD.
182
Table A.2 Effect of inhibitors on the ssDNA annealing and DNA pairing activities of RAD52 and on the DNA pairing activity of RAD51
ssDNA annealing Inhibition of DNA Inhibition of (Fluorescence pairing (D-loop RAD51 paring Inhibitors quenching), formation), (D-loop IC50, μM IC50, μM formation), %* D-A13 5.2 13.6±0.64 102.5±1.1 D-A19 4.8 7.2±0.28 93.7±2.1 D-A21 9.8 16.2±0.42 97.5±2.5 D-C17 6.0 17.6±0.78 92.5±5.0 D-C19 2.0 4.3±0.35 73.6± 2.2 D-E05 1.7 11.3±0.35 110.9±4.3 D-G09 2.0 14.8±2.47 108.2±3.1 D-G11 6.0 8.9±1.6 110.7±0.2 D-G23 5.6 7.2±0.96 75.6±7.1 D-I01 3.6 15.4±0.57 112.5±4.2 D-I03 5.0 8.0±1.7 95.5±4.3 D-I05 4.3 8.8±0.42 75.1±4.8 D-I07 2.0 2.7±0.35 38.4±6.4 D-I09 6.8 10.6±1.4 58.6±2.9 D-I11 4.1 6.7±0.78 48.6±2.0 D-I19 3.5 4.1±0.14 35.3±5.8 D-K17 2.9 4.8±0.85 89.7±0.4
Note:* The effect of the inhibitors on RAD51 DNA pairing activity was measured at the concentrations that correspond their 10 x IC50 for RAD52 pairing activity. 100% of the D-loop yield correspond to the extent of reaction in the presence of 4% DMSO; the actual extent of D-loops was 44.7±0.2 % under these conditions.
183
Then, we examined the selectivity of the RAD52 inhibitors using RAD51 as a non-specific target. RAD51 is structurally unrelated to RAD52 but shares DNA pairing activity with RAD52. Using the D-loop assay, we found that at concentrations 10-fold higher than the IC50 for RAD52 DNA pairing activity, none
of the 17 tested compounds (Figure A.5) showed a significant inhibition of D-loop
formation (Figure A.4D). However, under suboptimal conditions for the D-loop
assay at a reduced Ca2+ concentration (1 mM) three tested N-Methyl thieno
pyrazoles compounds, D-I07, D-I11, and D-I19 (Figure A.5), showed non-specific
inhibition of RAD51 greater than 2-fold relative to the DMSO control (Figure A.6;
Table A.2). Overall, as a result of the HTS and several confirmatory and selectivity
assays, we identified 14 specific inhibitors of ssDNA annealing and ssDNA pairing
activity of RAD52 in vitro.
184
Figure A.5 Structures of RAD52 inhibitors.
185
Figure A.6 The effect of the RAD52 inhibitors on DNA pairing activity of RAD51 in the D-loop assay. To form RAD51 nucleoprotein filament, RAD51 (1 μM) was incubated with 32P-labeled ssDNA (3 μM, nt) in buffer containing 25 mM Tris-Acetate, pH 7.5, 100 μg. ml-1 BSA, 1 mM calcium chloride, 1 mM ATP and 2 mM DTT for 15 min at 37 °C. Then inhibitors were added at the concentrations that correspond to their 10 x IC50 values for RAD52 pairing activity (see Figure 2C; Supplementary Table 2) and incubation continued for 15 min at 37 °C. D-loop formation was initiated by addition of supercoiled pUC19 DNA (50 μM, nucleotides) and was carried out 15 min at 37 °C. The reactions were stopped and the products processed as described in Materials and Methods. Error bars indicate SD.
A.4.2 Analysis of RAD52 inhibitors in human BRCA1- and BRCA2-deficient cells.
Since it was previously shown that inactivation of RAD52 by siRNA and peptide aptamer causes lethality of BRCA1- and BRCA2-deficient cells (Cramer-
Morales et al., 2013; Lok et al., 2013), we suggested that the RAD52 inhibitors can also inhibit the growth of BRCA1/2-deficient cells. First, we tested the effect of 14
RAD52 inhibitors identified in the biochemical assays on the growth of BRCA2- deficient Capan-1 cells and BRCA2-proficient BxPC3 cells. BxPC3 cells are commonly used as a control for Capan-1 cells; these two well-differentiated
186 pancreatic adenocarcinoma cell lines show significant similarity and share the mutation status of a number of tumor suppressor genes, including p53, p16, Rb, have strong expression of COX-2 and MMP-9 (Abbott et al., 1998; Bogliolo et al.,
2000; Yuan et al., 1999). We found that five of the tested inhibitors preferentially suppressed the growth of Capan-1 cells (Figure A.7A).
187
Figure A.7 Effect of the RAD52 inhibitors on cell survival (A) Capan-1 (BRCA2-) and BxPC3 (BRCA2+) cells and (B) on UWB1.289 (BRCA1-) and [UWB1.289 (+BRCA1)] (BRCA1+) cells. Capan-1 and UWB1.289 are denoted by the red line; BxPC3 and UWB1.289 (+BRCA1) indicated by a blue line. The experiments were repeated at least three times, error bars indicate SD.
188
We then tested the effect of RAD52 inhibitors on the growth of BRCA1- deficient (UWB1.289 BRCA1-) and BRCA1-proficient (UWB1.289 BRCA+) cells.
We found that 7 out 14 tested RAD52 inhibitors preferentially suppressed the growth of BRCA1-deficient cells; importantly, these 7 compounds included the 5 compounds that inhibited the growth of BRCA2-deficient cells (Figure A.7B). We also tested the effect of the 14 RAD52 inhibitors on the survival of another BRCA1- deficient cells, MDA-MB-436. Three out 14 compounds, which also inhibited the growth of UWB1.289 cells, showed an inhibitory effect on these cells compared with the isogenic MDA-MB-436 (BRCA1+) cells (Figure A.8A). Finally, we tested the effect of D-I03, the strongest inhibitor on BRCA1- and BRCA2-deficient cells, on BCR-ABL1 –positive BRCA1-deficient chronic myeloid leukemia (CML) patient cells (Podszywalow-Bartnicka et al., 2014). BRCA1-deficiency in these cells is due to constitutive downregulation of this protein. We found that D-I03 selectively diminished the growth potential of BRCA1-deficient CML patient cells in comparison to BRCA1-proficient normal counterparts (Figure A.8B).
189
Figure A.8 Effect of RAD52 inhibitors on the survival of MDA-MB-436 (BRCA1-) and MDA-MB-436 (BRCA1+) cells (A) and on the survival of on BCR- ABL1 –positive BRCA1-deficient CML cells and their BRCA1-proficient normal counterparts (B). The experiments were repeated at least three times, error bars indicate SD.
Overall, two compounds, D-G09 and D-I03, showed an inhibitory effect on all three tested BRCA1- and BRCA2-deficient cell lines. Importantly, these compounds showed significant structural similarity sharing the quinoline core
(Figure A.5). Another member of this structural group, D-G11, showed activity on two of the tested cell lines (UWB1.289 and Capan-1). Remarkably, three other compounds, D-G23, D-I05, and D-K17, that preferentially inhibited at least two
BRCA1/2-defficient cell lines also share a similarity, but belong to another structural type with the quinazoline core. From all tested compounds, D-I03 showed the strongest and most consistent inhibitory effect on all tested BRCA1-/-
190 and BRCA2-/- cell lines; moreover, it selectively inhibited the growth of BRCA1- deficient CML cells from patients.
A.4.3 Measurement of inhibitors binding to RAD52 by SPR.
We then tested whether RAD52 inhibitors that showed activity in the biochemical and cell-based assays physically interact with RAD52. Using the surface plasmon resonance (SPR) method, we demonstrated that D-I03 (3.12−50
μM) and D-G23 (3.125−25 μM) bind directly to RAD52 (Figure A.9). The anti-HIV mAb 2F5 structurally unrelated to RAD52 was used in control and the non-specific binding signal was subtracted from the RAD52 binding signal. The Kd values for
D-I03 and D-G23 are 26.1±4.5 μM and 34.0 ±8.9 μM, respectively. These values are somewhat higher than one might expect from the IC50 values in the biochemical and cell-based assays with these inhibitors. Because the active form of RAD52 is a hexamer, the apparent differences in the activities of inhibitors may suggest that inhibition of RAD52 requires only partial saturation of the RAD52 hexamer with inhibitors. Using the acridine orange displacement assay we showed that D-I03 and D-G23 did not bind DNA. Thus, these compounds inhibit DNA annealing and pairing activities of RAD52 through direct binding, not through interaction with DNA substrates.
191
Figure A.9 Measurement of D-I03 (A) and D-G23 (B) binding to RAD52. Compound D-I03 at concentrations of 3.125, 6.25, 12.5, 25, and 50 µM or compound D-G23 at concentrations of 1.56, 3.125, 6.25, 12.5, and 25 were injected to the chip with immobilized RAD52. Colored lines indicate experimental data, whereas black lines indicate fitting to the simple 1:1 binding model using the
192
ProteOn Manager Software version 3.0 (Bio-Rad). For D-G23 binding to RAD52, kinetic values are as follows: ka = 1.15 (±0.44) × 104 M-1s-1; kd = 3.62 (±0.7) × 10- 1 s-1; Kd = 34.0±8.9 µM. (C) For D-I03, the data did not fit to any available binding models and the Kd was determined using equilibrium analysis by plotting the response at equilibrium (Req) versus concentration of the compound. Experiments were repeated at least three times; error bars indicate S.D.
A.4.4 Inhibitors disrupt RAD52, but not RAD51, foci formation.
We wished to test whether D-I03 and D-G23 can inhibit RAD52 activities in
the cell. In response to DNA damage, RAD52 accumulates in the nucleus forming
distinct structures known as foci (Essers et al., 2002b; Liu and Maizels, 2000). It is
thought that the foci represent RAD52 complexes with DNA repair intermediates.
Inhibitors of RAD52 may decrease RAD52 foci formation by disrupting its interaction with DNA substrates. Indeed, we found that both D-I03 and D-G23
inhibited RAD52 foci formation induced by cisplatin in a BCR-ABL1-positive
BRCA1-deficient 32Dcl3 murine hematopoietic cell line that expresses GFP-
RAD52 (Cramer-Morales et al., 2013) (Figure A.10A). In the presence of D-I03 (2.5
μM) and D-G23 (2.5 μM), the fraction of cells with RAD52 foci (≥5 foci) was decreased approximately 2.0 - 2.5-fold, from 38.7 ± 10% to 17 ± 1% and 19 ±
0.4%, respectively; at the same time, the fraction of cisplatin-treated cells without foci was increased from 48.4 ± 6.2% to 71.9 ± 4.1 % and 57.6 ± 0.5 % (Figure
A.10A, bottom right panel). Thus, compounds of two chemotypes, represented by
D-I03 and G-23, were identified that inhibited the biochemical activities of RAD52
in vitro, showed preference in suppressing survival of BRCA1- and BRCA2-
deficient cells and inhibited the formation of damage-induced RAD52 foci
193 formation. We also tested a non-specific effect of D-I03, a strongest of theRAD52 inhibitors, on RAD51 foci formation. Using parental 32Dcl3 (BRCA1-proficient) cells, we found that D-I03 does not have a significant effect on RAD51 foci induced by cisplatin (Figure A.10B). Also, D-I03 alone induce neither RAD51 foci nor
RAD52 foci (in BRCA1-deficient cells; Figure A.10A) indicating low genotoxicity of this compound.
194
Figure A.10 Effect of RAD52 inhibitors D-G23 and D-I03 (2.5 µM) on GFP- RAD52 foci formation in response to cisplatin (10 µM) treatment. (A) GFP- RAD52 was constitutively expressed in p210BCR-ABL1-positive 32Dcl3 murine hematopoietic (BRCA1-deficient) cells. In control, cells were treated with D-I03 (2.5 µM) alone. On the bottom right, the data are shown as a graph. Cells with a small foci number (1-5) were excluded from calculations. Error bars represent SD. (B) Effect of D-I03 (2.5 µM) on RAD51 foci formation with and without cisplatin (10 µM)
195 treatment in parental 32Dcl3 (BRCA-proficient) cells. On right, the data are shown as a graph. Average numbers of foci per cell on right panel were determined in cells with more than 10 foci. Error bars represent SD.
A.4.5 D-I03 inhibits single-strand DNA annealing, but not gene conversion in
U2OS cells.
In both yeast and mammalian cells, RAD52 promotes single-strand DNA
annealing (Krogh and Symington, 2004; Stark et al., 2004). Single-strand
annealing is a type of homologous recombination that is initiated at double-strand
breaks and mediated by annealing of fortuitous direct repeats flanking double-
strand break ends after their exonucleolytic resection. This mechanism leads to
end re-joining with concomitant deletion of sequences between direct repeats.
Here, using the SA-GFP construct integrated into the chromosomal DNA (Gunn et al., 2011; Gunn and Stark, 2012), we wished to examine the effect of RAD52 inhibitor D-I03 on single-strand annealing in U2OS cells. The SA-GFP reporter system contains a 5’ fragment of the GFP (5’-GFP) gene, and a 3’ fragment of the
GFP (3’-GFP) that contains an 18-bp I-SceI site (Figure A.11A). The GFP fragments are separated by 2.7 kb region and share a 266 bp region of homology.
Transfection of cells with I-SceI expressing vector pCBASce induces a double- strand break in the 3’-GFP. Repair of the double-strand break by single-strand annealing leads to the formation of GFP+ cells. Thus, each single-strand annealing event can be scored by the appearance of a green fluorescent cell.
196
Figure A.11 D-I03 inhibits single-strand annealing, but not homology dependent recombination (gene conversion) in U2OS cells. (A) The scheme of the reporter systems. The SSA-GFP reporter contains a 5’ fragment of the GFP (5’-GFP) gene, and a 3’ fragment of the GFP (3’-GFP) with an I-SceI site. The HDR-GFP reporter system contains the GFP gene interrupted by a Sce-I site and a fragment of the GFP with truncated 3’- and 5’-rerminus. Repair of the I-SceI- induced double-strand break by either single-strand annealing or gene conversion leads to the formation of GFP+ cells (21). Effect of D-I03 on the repair of the I- SceI-induced double-strand breaks in U2OS cells carrying the chromosomally located SSA-GFP (B) or HDR-GFP (C) reporter is shown by red line. In a control,
197 the effect of D-I03 on formation GFP+ cells was measured after transfection of U2OS cells with a pMX-GFP plasmid expressing GFP protein (green line). Formation of GFP+ cells in the absence of D-I03 was expressed as 100%. Experiments were repeated at least three times; error bars indicate S.D.
We found that D-I03 reduces the formation of GFP+ cells in a concentration dependent manner; at 30 μM D-I03 the yield of GFP+ cells was reduced approximately 3.4-fold (Figure A.11B, red line; Figure A.12). In control, we measured the effect of D-I03 on the formation of GFP+ cells when U2OS cells were transfected with pMX-GFP plasmid encoding GFP (Figure A.11B; green line).
We found that up to 30 μM, D-I03 had no significant effect on the recovery of GFP+ cells.
198
Figure A.12 The effect of D-I03 on the repair of the I-SceI-induced double- strand breaks in U2OS cells carrying the SSA-GFP reporter was determined by flow cytometry. Green fluorescence (GRN-Hlog) was plotted against red fluorescence (RED-Hlog) for the sample of 10,000 cells. The GFP+ population is denoted by the elliptical M1 marker. Cells with I-SceI-induced double-strand
199 breaks were treated with D-I03 at 0, 5, 10, 15, 20, or 30 μM (panels 1−6, respectively). In a negative control, U2OS cells were transfected with pUC19, instead of pCBASce that expresses I-SceI (panel 7). U2OS cells transfected with a pMX-GFP plasmid expressing GFP protein were used as a positive control (panel 8).
We then tested the effect of D-I03 on double-strand break repair via the canonical homology dependent recombination (also known as gene conversion) mechanism using the chromosomally integrated DR-GFP construct in U2OS cells
(Gunn et al., 2011; Gunn and Stark, 2012). Previously, it was shown that RAD52 has no significant effect on the homology-dependent recombination in mammalian cells (Stark et al., 2004). The DR-GFP construct consists of two inactive copies of the GFP gene, one that is disabled by insertion of I-SceI recognition site and another (iGFP) is truncated at both ends (Figure A.11A). A unique double-strand break is generated in this construct after the cells are transfected with the pCBASce plasmid. The repair of this double-strand break by gene conversion using iGFP as a template gives rise to the functional GFP gene. We found that D-
I03 does not have a significant effect on the formation of GFP-positive cells (Figure
A.11C, red line; Figure A.13). D-I03 also has no effect on GFP expression or on the recovery of GFP-positive cells when U2OS cells were transfected with pMX-
GFP plasmid encoding GFP (Figure A.11C, green line). Taken together, our results indicate that consistent with inhibition of RAD52 in human cells D-I03 reduces the level of double-strand break-induced single-strand annealing, but not homology- dependent recombination.
200
Figure A.13 The effect of D-I03 on the repair of the I-SceI-induced double- strand breaks in U2OS cells carrying the HDR-GFP reporter (to measure gene conversion) was determined by flow cytometry. Green fluorescence (GRN- Hlog) was plotted against red fluorescence (RED-Hlog) for the sample of 10,000
201 cells. The GFP+ population is denoted by the elliptical M1 marker. Cells with I- SceI-induced double-strand breaks were treated with DI03 at 0, 5, 10, 15, 20, or 30 μM (panels 1−6, respectively). In a negative control, U2OS cells were transfected with pUC19, instead of pCBASce (panel 7). U2OS cells transfected with a pMX-GFP plasmid expressing GFP protein were used as a positive control (panel 8).
A.5 DISCUSSION
RAD52 is an evolutionarily conserved eukaryotic protein, a member of the
homologous recombination pathway that is responsible for repair of double-strand breaks, recovery of stalled replication forks, and faithful chromosome segregation during meiosis(Kowalczykowski, 2000; Moynahan and Jasin, 2010; Symington and Gautier, 2011; van Gent et al., 2001). In yeast, Rad52 plays a key role in
homologous recombination; Rad52 null mutation obliterates nearly all types of
recombination events and renders cells extremely sensitive to double-strand
break-inducing agents. Surprisingly, in mice RAD52 knockouts show virtually no
DNA repair phenotype. The recent discovery that RAD52 mutations are synthetically lethal with mutations in BRCA1/2, PALB2, and RAD51 paralogs indicated that in mammalian cells RAD52 constitutes an alternative homologous
recombination sub-pathway that may play a back-up role during double-strand
break repair (Feng et al., 2011; Lok et al., 2013). This independent/redundant role
of RAD52 in mammalian homologous recombination is also supported by
cytological data. In response to DNA damage both RAD51 and RAD52 form
nuclear foci, which are thought to play a role of the DNA repair centers. However,
foci formation by these proteins show only partially overlap(Liu and Maizels, 2000)
202 and their formation is differently affected by mutations in other homologous recombination genes; whereas RAD51 foci formation depends on the functional
BRCA1, BRCA2, and RAD51 paralogs, RAD52 foci formation is independent of these proteins(van Veelen et al., 2005).
The mechanistic basis for the RAD52 function in homologous recombination in mammalian cells remains to be elucidated. Biochemical studies indicate that in S. cerevisiae Rad52 may play a role of a mediator that helps to load
Rad51 recombinase on ssDNA at the site of double-strand breaks overcoming an inhibitory effect of Replicative Protein A (RPA), an abundant protein that has high affinity for ssDNA (Sugiyama and Kowalczykowski, 2002; Sung, 1997). The
RAD51 nucleoprotein filament formed on ssDNA then searches for homologous dsDNA and invades it to form joint molecules (D-loops) that provide a template and a primer to recover the DNA lost at the site of the double-strand break.
However, the mediator activity was not demonstrated for human RAD52 in vitro
(Jensen et al., 2010).
RAD52 possesses ssDNA annealing activity both in vitro and in vivo which can contribute to homologous recombination in several different ways (Mortensen et al., 1996; Stark et al., 2004; Sugiyama et al., 1998). This activity was implicated in the second DNA end capture during RAD51-dependent double-strand break repair resulted in the formation of double D-loops and then of Holliday junctions
(Bugreev et al., 2007; McIlwraith and West, 2008; Sugiyama et al., 2006). It was also proposed that the ssDNA annealing activity of RAD52 may be responsible for a step that follows DNA repair synthesis and D-loop dissociation: re-annealing of
203 the displaced ssDNA with the second DNA end of the double-strand break
(Bugreev et al., 2007). In addition, RAD52 ssDNA annealing activity may also contribute to homologous recombination in an RAD51-independent manner.
Genetic data indicate that this activity may be responsible for the error-prone DNA single-strand annealing sub-pathway of homologous recombination, which is independent of RAD51 (Pâques and Haber, 1999). Furthermore, biochemical data show that RAD52, similar to RAD51, is able to promote D-loop formation, albeit with lower efficiency (Kagawa et al., 2008), suggesting that RAD52 may potentially substitute RAD51 in some homologous recombination events.
Importantly, the RAD52-dependent mechanism of double-strand break repair is essential for viability in mammalian cells that are defective in BRCA1,
BRCA2, PALB2, or in five RAD51 paralogs (Feng et al., 2011; Lok et al., 2013).
Synthetic lethality provides a conceptual framework for discovering drugs that selectively kill cancer cells while sparing normal tissues (Bryant et al., 2005;
Farmer et al., 2005; Helleday et al., 2007). In the current paper, we exploited synthetic lethality between the RAD52 and BRCA1&2 genes (Feng et al., 2011;
Lok et al., 2013). We proposed that targeting of RAD52 with small molecule inhibitors will disrupt the RAD52-dependent homologous recombination sub- pathway in BRCA1- and BRCA2-deficient cells causing their lethality. The data with peptide aptamer that disrupts RAD52 binding to ssDNA supported this hypothesis (Cramer-Morales et al., 2013). RAD52 represents an attractive potential therapeutic target also because no RAD52 mutations or inactivation has been documented in human tumors (Lok et al., 2013). Using HTS, we identified
204 several small molecule compounds that specifically inhibit RAD52 ssDNA annealing and DNA pairing activities. Importantly, the selected inhibitors of two different chemotypes showed inhibitory effect on tested BRCA1- and BRCA2- deficient cells. The compound, D-I03, with the strongest inhibitory effect in human cells also selectively inhibited the growth of CML patient cells, in which the expression of BRCA1 was constitutively reduced (Podszywalow-Bartnicka et al.,
2014). Finally, RAD52 inhibitors D-I03 and, to a smaller extent, D-G23 also disrupted the formation of RAD52 foci induced by cisplatin. At the same time, D-
I03 had no significant effect on the formation of cisplatin-induced RAD51 foci indicating specific targeting of RAD52 in cells. In addition, D-I03 seems to have a low genotoxicity, as it did not induce RAD52 or RAD51 foci in BRCA1-defecient or
BRCA1-proficient cells, respectively. In accord with specific targeting RAD52 in human cells, D-I03 inhibited the single-strand annealing in USO2 cells, without significantly affecting canonical homology-dependent recombination. Previously, genetic experiments demonstrated that in mammalian cells RAD52 plays an important role in single-strand annealing, but not in homology-dependent recombination (Stark et al., 2004).
Structurally six RAD52 inhibitors that show the biological effect in human cells belong to two scaffolds, the quinoline, and quinazoline (Figure A.5). These scaffolds represent attractive starting points for medicinal chemistry optimization as at least three distinct regions of these chemotypes can be readily modified.
Another important aspect to evaluate in prospective chemotypes is their calculated property profile. Gratifyingly, both chemotypes, represented by D-I03 and D-G23,
205 have calculated properties within ranges generally considered “drug-like” and favorable for important parameters such as oral bioavailability (Table A.3).
Table A.3. Calculated properties of D-I03 and D-G23 compounds*
D-I03 D-G23
molecular weight 429 354 cLogP 3.65 3.29 TPSA 47 89 HBD 2 3 HBA 5 7 rotatable bonds 7 7 * calculated using ADRIANA.code
TPSA = topological polar surface area HBD = hydrogen bond donors HBA = hydrogen bond acceptors
(https://www.molecular-
networks.com/files/docs/adrianacode/adrianacode_flyer.pdf)
Previously, it was demonstrated that the N’-terminal domain is responsible
for ssDNA annealing and DNA pairing activities of RAD52 (Kagawa et al., 2002).
Therefore, we suggest that the selected compounds may specifically target this
domain. However, more work is needed to investigate the mechanism of action of
the inhibitors. We propose that RAD52 inhibitors can be used as prototypes for
development of novel therapies against hereditary breast cancer and ovarian
206 cancers with defective BRCA1 or BRCA2 proteins. This therapy can also be potentially applied to sporadic cancers in which BRCA1 or BRCA2 are either mutated or downregulated, e.g., due to constitutive promoter methylation (Esteller,
2008; Hansmann et al., 2012). Specific RAD52 inhibitors can also be used as a research tool to study the RAD52 biochemical activities and cellular functions.