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The Influence of Chaperones and

Histone Modifying Proteins in Regulating the DNA Damage Checkpoint

Senior Thesis

Presented to The Faculty of the School of Arts and Sciences Brandeis University

Undergraduate Program in Biology James Haber, Advisor

In partial fulfillment of the requirements for the degree of Bachelor of Science

By Lizabeth J. Katsnelson

April 2015

Copyright by Lizabeth J. Katsnelson

Committee members:

Name: Dr. James E. Haber Signature: ______

Name: Dr. Susan Lovett Signature: ______

Name: Dr. James Morris Signature: ______

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Acknowledgments

People say, “It takes a village to raise a child” and just like raising a child, it takes a village to finish a senior thesis. There are so many people I would like to thank for helping and supporting me throughout this process. First, I would like to thank my advisor, Dr. James Haber, for allowing me to work on this project in his laboratory and for believing in my skills as a young scientist. I would also like to thank the rest of the members of my thesis committee, Dr. Susan Lovett and Dr. James Morris, for reading and assessing my thesis.

This project and thesis would have never come together without the extraordinary support from my mentor, Michael Tsabar. Michael’s mentorship was an invaluable aspect to my growth as a researcher and I am truly grateful to have had him as my guide during this journey. I would also like to thank all the other members of the Haber lab, both past and current, who were always willing to provide guidance to me and all the other undergraduates in the lab.

Lastly, I would like to thank my friends and family for all their support. I could not have accomplished any of this with out the love and support of both my parents, my grandmother, and my sister, who all encouraged me to go to Brandeis and always believed in my abilities.

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Abstract

DNA double stranded breaks (DSBs) occur constantly, and are highly deleterious, which is why eukaryotic cells have evolved mechanisms of repair to ensure genomic stability. A crucial aspect of the DNA damage response is recognition of the lesion and activation of the DNA damage checkpoint, which prevents cells from dividing prior to repairing the lesion. Following a DSB in yeast, the two PI3-like protein kinases Mec1 and

Tel1 phosphorylate a cascade of downstream effectors that lead to cell cycle arrest. A key target for Mec1 regulated is Rad53, which, following Mec1 mediated phosphorylation undergoes autophosphorylation. Following repair, cells must properly deactivate the checkpoint in order to continue the cell cycle, a process termed recovery.

Recovery defective cells fail to deactivate the checkpoint and therefore cannot resume cell cycle progression, despite being able to repair. This study focuses on the role of histone chaperones and histone modifying proteins in the deactivation of the checkpoint, specifically Asf1, CAF-1, Rtt109, and Rtt101. Asf1 and CAF-1 are -H4 chaperones. Interestingly, Asf1 binds Rad53 prior to suffering damage. Once Rad53 is phosphorylated during the damage response, its affinity to Asf1 is greatly reduced.

Rtt109 is a histone acetyl transferase and acetylates H3K56 after Asf1 binds H3, which then weakens Asf1-H3 interactions. Rtt101, a ligase, ubiquitylates H3K56ac to aide in the dissociation of Asf1 from H3. Therefore, the hypothesis proposed in this thesis is that dissociating H3-H4 from Asf1 is necessary to allow Asf1 to interact and sequester

Rad53 following repair, thus deactivating the checkpoint and facilitating recovery.

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Introduction

The nucleus of eukaryotic cells holds genomic information in the form of , which is formed by repetitions of its functional unit, the (Polo,

2015). Each nucleosome protects approximately 146 base pairs of DNA that wrap around a histone octamer. The histone octamer contains a (H3-H4)2 tetramer and two

H2A-H2B dimers (Burgess and Zhang, 2013). Depositing H3-H4 molecules is the rate- limiting step in the formation of (Han, et al., 2013). Nucleosomes pose a barrier for DNA-related processes such as replication, repair, or transcription. In order to modify nucleosome affinity to DNA, can be post-translationally modified by , , phosphorylation, and ubiquitylation to stimulate or disable certain cellular processes (Burgess and Zhang, 2013).

DNA damage can result from many exogenous and endogenous sources such as radiation, reactive oxygen species, or replication across nicked DNA (Harrison and

Haber, 2006). Double-strand breaks (DSBs) are the most detrimental form of DNA damage and failure to repair DSBs can result in genomic instability conducive to the development of many diseases, including cancer (Finn, et al., 2012). Eukaryotic cells have elaborate and highly conserved pathways of DNA damage sensing, signaling, and repair termed the DNA damage response (DDR) (Bradbury and Jackson, 2003). Not only does the DDR require recognition of the altered DNA structure, but also the recruitment of repair factors in order to initiate repair. Once the cell has repaired damaged DNA, the chromatin structure of the repaired DNA must be reassembled for the cell to return to its normal cell cycle (Hu et al., 2001). Failure to repair DSBs can result in cell death

(apoptosis) or in the propagation of mutations that can lead to cancer. Even a single DSB 5 can lead to apoptosis, directly inactivate key genes, chromosomal translocations, or generate unstable chromosomal abnormalities, making the repair response very important

(Bradbury and Jackson, 2003). These pathways are highly conserved from budding yeast

(Saccharomyces cerevisiae) to humans, which is why significant findings in yeast may be extrapolated to vertebrates (Finn, et al., 2012).

The DNA damage checkpoint is central to the DDR. When activated by damage, the cell cycle arrests to allow for proper repair (Finn, et al., 2012). In yeast, cell cycle arrest occurs at the G2/M checkpoint prior to anaphase and damaged DNA must be repaired before the cell can enter mitosis (Kim and Haber, 2009, Harrison and Haber,

2006). Although the DDR is highly conserved from yeast to humans, mammalian cells mainly arrest the cell cycle at the G1/S checkpoint, which is before DNA is replicated

(Price and D’Andrea, 2013). The proteins involved in turning on, maintaining, and turning off the checkpoint are named checkpoint proteins. Checkpoint proteins were originally identified from experiments in which their loss of function caused defects in cell cycle progression or cell cycle arrest in response to exposure to DNA damaging agents (Branzei and Foiani, 2005). The checkpoint response acts as a signal transduction cascade in which sensor proteins first detect specific structures or lesions in the DNA.

Then, protein kinases activate the transducers, transmit, and perhaps also amplify the checkpoint signal to the receivers. The receivers, which are essentially effectors, are downstream targets of the initial checkpoint. This includes the DNA repair apparatus and the cell cycle machinery responsible for modulating cell cycle progression in response to

DNA damage (Branzei and Foiani, 2005). These processes are accompanied by alterations in chromatin structure by various kinases, histones, histone chaperones, 6 nucleosomes, and others (Tsabar and Haber, 2013).

Eukaryotes respond to DNA damage by repairing through two major pathways called homologous recombination (HR) or non-homologous end-joining (NHEJ)

(Bradbury and Jackson, 2003). In HR, after suffering a DSB, the DNA ends are processed in a 5’ to 3’ direction in a process termed resection, producing RPA bound 3’ single- stranded tails. In gene conversion (GC), one type of HR, the resected DNA ends recognize and pair with homologous sequences from a sister chromatid, an allelic locus, or at an ectopic location in the genome termed “donor” (Mehta and Haber, 2014). A filament of the Rad51 recombinase that forms on the resected ends facilitates the search for homology and pairing with homologous sequences. Binding of Rad51 is facilitated by

Rad54 and Rad52, which are recruited to the DNA ends. Rad51 then mediates the search for homologous sequences and then once it is found, Rad51 catalyzes the reaction in which the damaged strand invades the donor. DNA polymerase extends the DNA from the 3’ terminus of the damaged molecule and the ends are ligated by DNA ligase I

(Jackson, 2001). Another form of HR is single-strand annealing (SSA), which results in the formation of a deletion between homologous sequences flanking a DSB. Rad51 is not required for this process because the homologous sequence is on the same chromosome, which is why this is a deleterious form of HR and is error prone (Scully and Xie, 2013).

Unlike HR, NHEJ requires little to no homology when ligating broken ends and normally results in small deletions or insertions. NHEJ can occur throughout the cell cycle, specifically in G1 when HR 5’ to 3’ resection of DSB ends is blocked (Mehta and

Haber, 2014). During NHEJ, there is usually limited nuclease digestion followed by ligation of the broken ends. This may result in loss of some genetic material, but it is the 7 predominant pathway for DSB repair in mammals because it is efficient in removing potentially lethal DSBs (Bradbury and Jackson, 2003). In this paper, we focus on HR because the yeast models used in this project repair DSBs via an ectopic GC event or a

SSA event.

Response to DNA damage and activating the checkpoint in eukaryotic cells depends greatly on members of the phosphatidylinositol 3’ kinase-like kinase (PI3KK) family (Harrison and Haber, 2006). Mammalian cells contain two important DNA damage checkpoint PI3KK’s, Ataxia telangiectasia mutated protein (ATM) and ataxia telangiectasia and Rad3-related protein (ATR). The Saccharomyces cerevisiae orthologs to ATM and ATR are Tel1 and Mec1, respectively. In humans, ATM is the protein that is mutated in ataxia telangiectasia, which is a radiosensitivity and genome instability disorder categorized by progressive cerebellar degeneration and increased cancer incidence (Bradbury and Jackson, 2003). Seckel Syndrome, a disorder characterized by proportionate growth retardation and severe microcephaly, is caused by mutations in human ATR (Gobbini et al., 2013). ATM and its yeast counterpart Tel1 are not as essential proteins as ATR and its yeast counterpart Mec1 (Bradbury and Jackson, 2003).

Mec1 is crucial in arresting the cell cycle before metaphase after a single unrepaired DSB is recognized (Harrison and Haber, 2006). Recruitment of Mec1 to a

DSB requires the presence of RPA coated ssDNA 3’ tails. Recognition of ssDNA coated with RPA requires Mec1 to be bound to Ddc2 (Gobbini et al., 2013). The 3’-ended ssDNA tails trigger the Mec1-Ddc2 complex to activate the checkpoint kinase cascade

(Harrison and Haber, 2006). Two of Mec1’s downstream targets, Chk1 and Rad53, are essential for arrest following DNA damage (Tsabar and Haber, 2013). Once 8 phosphorylated by Mec1, Rad53 associates with Mec1-phosphorylated Rad9 scaffold protein and can then autophosphorylate (Tsabar and Haber, 2013). The Rad53 human ortholog, Chk2, is a tumor suppressor (Harrison and Haber, 2006 and Stolz et al, 2011).

Another key part of the Mec1 pathway is the 9-1-1 clamp, which is composed of Ddc1,

Rad17, and Mec3. Mec1-Ddc2 and the 9-1-1 complex at sites of damage directly stimulate Mec1 kinase activity (Gobbini et al., 2013). Deletion of Mec1, Ddc2, or Rad53 in yeast strains is lethal (Harrison and Haber, 2006).

Tel1 is activated by blunt-ended DNA bound by the MRX complex, which contains Mre11, Rad50, and Xrs2 (Harrison and Haber, 2006). MRX and another key protein, Sae2, initiate DSB resection. MRX and Sae2 work together to remove oligonucleotides from the 5’ ends of the break, which creates 3’-ended ssDNA tails that then undergo resection (Cannavo and Cejka, 2014). Although Mec1 is responsible for activation of the checkpoint after damage, it has been shown that Mec1 and Tel1 play redundant roles in controlling DNA repair by phosphorylating the necessary DNA repair proteins (Tsabar and Haber, 2013).

Mating type switching of Saccharomyces cerevisiae is a useful genetic tool used in many types of experiments. Mating type is determined by two nonhomologous alleles,

MATa and MATα. Mating type switching occurs by site-specific homologous recombination in which one MAT allele is replaced by the opposite MAT allele (Haber,

2012). MAT switching involves two copies of mating-type information at the heterochromatic loci, HMLα and HMRa, which are located at opposite ends of the same chromosome encoding MAT, chromosome III (Haber, 2012; Kim and Haber, 2009).

Studying MAT switching has generated insights into the control of cell lineage, silencing 9 gene expression, and regulation of availability of donor sequences and especially the mechanisms of double-strand break repair (Haber, 2012).

Another useful genetic tool is the HO endonuclease, which is used in various strains to induce DSBs. HO has three possible targets in a haploid cell: MAT locus,

HMLα, and HMRa. Because only the MAT locus is accessible under normal conditions, there is a single, programmed DSB on the MAT locus only in parent cells, prior to the initiation of DNA replication (Haber, 2012). In the very well known yeast strain, YJK17, galactose induces the HO endonuclease to produce a DSB at the MATα locus on chromosome III. Because the strain lacks both HML and HMR, the break cannot be repaired via intrachromosomal HR, which is how they normally switch mating type.

These cells contain a homologous donor, MATa-inc, on chromosome V, which repairs the

DSB on chromosome III via ectopic gene conversion (GC). The “a-inc” sequence is a mutated HO recognition site meaning it cannot be cleaved by HO endonuclease and therefore only the MAT sequence on chromosome III will be cleaved (Kim and Haber,

2009).

MAT switching induced by galactose-regulated HO endonuclease is a fairly slow process, which normally require about an hour to complete (Haber, 2012). Having a single unrepaired HO-induced DSB is enough to cause a prolonged Mec1 dependent cell cycle arrest in G2/M that can last about 12-14 hours. When there are two unrepairable

DSBs present, the checkpoint is even stronger and cells can become permanently arrested

(Harrison and Haber, 2006).

DSB repair can be highly influenced by histone modifications and ATP- dependent chromatin remodeling reactions (Gobbini et al., 2013). For example, Mec1 10 and Tel1 phosphorylate serine 129 of the histone variant H2AX during DNA damage repair. Phosphorylated H2AX (termed γ-H2AX) is detected soon after a DSB forms. γ-

H2AX is normally found over a large region of chromatin flanking the DSB. γ-H2AX has been shown to contribute to DNA repair and also plays a conserved role in the DNA damage checkpoint. It has been suggested that one of the most important roles of γ-

H2AX is maintaining the checkpoint because strains that lack γ-H2AX activated the checkpoint normally but prematurely deactivated it (Harrison and Haber, 2006).

The assembly and disassembly of nucleosomes is crucial for cell cycle regulation and genome stability (Koning et al., 2007). Therefore, specific proteins called histone chaperones and ATP-dependent chromatin remodelers assist in nucleosome maintenance.

ATP-dependent chromatin remodelers catalyze the disruption of DNA-histone interaction via ATP hydrolysis (Lans et al., 2012). Histone chaperones are defined as proteins that associate with histones and are involved in their transfer but are not necessarily part of the final product and they are classified according to which histones they bind to (Koning et al., 2007). Histone chaperones play critical roles during DNA replication, transcription, and in the DNA damage response. During replication, histone chaperones disrupt chromatin organization and then restore it after fork passage (Gurard-Levin et al., 2014).

During transcription, histone chaperones rearrange chromatin structure in order for RNA polymerase II machinery to progress through the genome (Gurard-Levin et al., 2014 and

Koning et al., 2007). During the DDR, histone chaperones are recruited after the phosphorylation of H2AX to displace histones around the break site. This allows for damage repair proteins to have access to the break site and properly repair the DSB

(Gurard-Levin et al., 2014). This paper will focus on histone chaperones involved in the 11

DDR and their roles in the regulation of the checkpoint.

An important histone , Chromatin Assembly Factor 1 (CAF-1), deposits

H3-H4 during DNA replication and is thought to be a key factor in the formation of (H3-

H4)2 tetramers (Yu et al., 2015). CAF-1 is known to promote nucleosome formation on newly synthesized DNA by depositing newly formed H3-H4 onto DNA (Gurard-Levin et al., 2014 and Burgess and Zhang, 2013). Yeast CAF-1 is composed of three subunits:

Cac1, Cac2, and Cac3 (Kim and Haber 2009). Cac1 is identified as the largest subunit of

CAF-1 and research has shown that deletion of the CAC1 gene has caused an increase in

Okazaki fragment length and sensitivity to DNA damage-inducing agents, specifically

UV (Burgess and Zhang, 2013 and Kim and Haber, 2009). Another important histone chaperone, Regulator of Ty transposition 106 (Rtt106), is said to have the same effect as

CAF1 in that they both assist in the formation of the (H3-H4)2 tetramer. While CAF1 and

Rtt106 can both assist in (H3-H4)2 formation, Rtt106 is the protein responsible for (H3-

H4)2 deposition on DNA at the replication fork (Burgess and Zhang, 2013).

Another key histone chaperone is the Anti-silencing Factor 1 (Asf1), which is the most conserved eukaryotic H3-H4 chaperone (Gurard-Levin et al., 2014). Asf1 binds H3-

H4 with similar affinity as CAF-1 or Rtt106 and has been shown to relay H3-H4 to CAF1 or Rtt106 to be transformed into (H3-H4)2 tetramers (Burgess and Zhang, 2013). Asf1 has also been noted to promote acetylation of H3 56 (H3K56ac) by interacting with the histone acetyl transferase, Rtt109 (Tyler et al., 2008). Acetylation of H3K56 has been suggested to be a signal for recovery and contributes towards chromatin assembly after

DSB repair (Kim and Haber, 2009 and Tyler et al., 2008). It has been noted that Asf1 and

H3K56ac are required for the efficient association of H3-H4 with Rtt106 and CAF-1 12

(Burgess and Zhang, 2013). Deletion of Asf1 causes hypersensitivity to DNA-damaging agents and can cause cells to accumulate in G2/M because the DNA damage checkpoint is activated, even though the cells can properly repair DSBs (Emili et al., 2001 and Kim and Haber, 2009).

Another crucial protein involved in maintaining genomic stability is the cullin,

Rtt101 (Han, et al., 2013). Cullins act as scaffolds for assembling multisubunit ubiquitin ligases. Cullins are characterized by a conserved C-terminal domain that interacts with the RING finger protein RBX1/ROC1/Hrt1, which recruits ubiquitin-conjugating (Zaidi et al., 2008). Rtt101 is a member of the cullin-RING finger E3 ubiquitin ligase family and it assists in the association of a multisubunit ubiquitin ligase, Rtt101-

Mms1, which is required for accurate replication through natural pause sites and damaged templates (Han, et al., 2013 and Zaidi et al., 2008). Mms1 is an adaptor protein for substrate binding and it shares sequence homology with human DDB1, which binds

CUL4A and CUL4B to form the Cul4-DDB1 E3 ligase, similar to Rtt101-Mms1. In mammalian cells, the CUL4-DDB1 complex ubiquitylates various proteins involved in

DNA replication and chromatin dynamics. Mutations of human Cul4A have been recognized in a variety of cancers (Han, et al., 2013).

Yeast cells lacking Rtt101 are sensitive to DNA-damaging agents, defective in replication through damaged DNA or natural pause sites, and exhibit defects in the deposition of H3K56ac onto replicating DNA. Rtt101 functions in the same pathway as

Asf1 and Rtt109 to maintain genomic stability. After H3 is acetylated at lysine 56 by

Rtt109, Rtt101-Mms1 E3 ubiquitin ligase binds and ubiquitylates H3K56ac-H4, which is proposed to weaken the Asf1-H3-H4 interaction and facilitates the transfer of H3-H4 13 from Asf1 to histone chaperones CAF-1 and Rtt106 (Zaidi et al., 2008). Mms1 is necessary for the ubiquitylation of H3K56ac-H4 because it mediates the interaction between Rtt101 and H3K56ac-H4. It has also been shown that Rtt101-Mms1 preferentially ubiquitylates acetylated H3-H4 (H3K56ac) over non-acetylated H3-H4

(Han, et al., 2013). Moreover, deleting RTT101 or mutating the H3 lysine 56 residue to arginine impairs nucleosome assembly and promotes Asf1-H3-H4 interactions. By enhancing the Asf1-H3-H4 interaction, less H3-H4 will be transferred to CAF-1 and

Rtt106 which will therefore result in less (H3-H4)2 availability for nucleosome formation.

In human cells lacking Cul4A or DDB1, an increase in Asf1a and Asf1b association with

H3-H4 occurs. This confirms that the yeast Rtt101-Mms1 and human Cul4A-DDB1 complexes are negative regulators of the Asf1-H3-H4 interactions (Han, et al., 2013).

During normal yeast cell growth, Rad53 exists in a stable complex with Asf1.

Rad53 and Asf1 dissociate during DNA damage as Rad53 is phosphorylated by Mec-1

(and then hyperphosphorylated by autophosphorylation) and does not bind Asf1 anymore while Asf1 binds H3-H4 (Emili et al., 2001 and Jiao et al., 2012). Therefore, in the absence of DNA damage, Rad53 competes with H3-H4 for Asf1 binding (Emili et al.,

2001 and Jiao et al., 2012). Rad53 is dephosphorylated by the protein 2C

(PP2C), Ptc2, and Ptc3, which leads to checkpoint inactivation (Guillemain et al., 2007). Along with checkpoint regulation, Rad53 is also involved in the degradation of excess histones that are not packaged into chromatin (Gunjan and Verreault, 2003).

This is also a key function of Rad53 because overexpression of histones has been shown to cause sensitivity to DNA damage (Gunjan and Verreault, 2003).

The term “checkpoint recovery” indicates that the DNA repair process has been 14 completed and checkpoint activation is downregulated while the term “adaptation” designates re-entry to the cell cycle despite failure to repair DNA damage (Branzei and

Foiani, 2006). In adaptation and recovery, cells inactivate upstream elements of the DNA damage checkpoint, which prevents effectors from blocking cell cycle progression. The dephosphorylation of Rad53 is a key event in both adaptation and recovery. The disappearance of phosphorylated Rad53 has been accompanied by adaptation and recovery, while adaptation and recovery defective mutants exhibit persistent Rad53 phosphorylation (Guillemain et al., 2007).

A previous study, (Kim and Haber, 2009), found that single deletion of ASF1 or

CAC1 in the YJK17 strain containing one galactose-induced and repairable DSB resulted in no recovery defects, but an ASF1 CAC1 double deletion resulted in a recovery defect.

However, in asf1Δcac1Δ both DSB repair and the formation of γH2AX occurred normally. Furthermore, the absence of Asf1 and/or CAF-1 did not impair the cell’s ability to adapt (Kim and Haber, 2009). In a study done after this, (Fiona Aguilar, Brandeis

University Senior Thesis, 2014), a strain constructed with two inducible DSBs was then mutated with asf1Δ, cac1Δ, asf1Δcac1Δ, rtt109Δ, or cac1Δrtt109Δ. In the strain with two

DSBs, it was shown that asf1Δ was recovery defective, while cac1Δ exhibited a WT phenotype and was therefore not recovery defective. YJK17 rtt109Δ was not recovery defective, but the strain containing two DSBs and rtt109Δ was recovery defective, displaying the same phenotype as asf1Δ. Strains containing cac1Δrtt109Δ also behaved like asf1Δ, recovering the checkpoint normally in the single DSB strain while being recovery defective in the two DSB strain. These results led us to believe that Asf1 and

Rtt109 had a more important function than CAF-1 in checkpoint recovery. 15

Given the data provided that deleting Asf1, CAF-1, Rtt109, or Rtt101 results in hypersensitivity to DNA damage, genomic instability, and lowered viability, the goal of this project was to see how histone chaperones interact with histones and checkpoint proteins in the recovery pathway. Our project focused on Asf1, CAF-1, Rtt101,

Rtt109, and Rad53 during the inactivation of the checkpoint and re-entry into the cell cycle. Since Asf1 will not bind phosphorylated Rad53, we hypothesize that the binding of Rad53 to Asf1, as

Rad53 is dephosphorylated by

PTC2, PTC3, and PPH3, prevents further Figure 1: Proposed model a) Asf1 binds to H3-H4 and Rad53 during normal cell cycling. Asf1 hands off H3- autophosphorylation and H4 to CAF-1 which deposits (H3-H4)2 tetramers onto newly synthesized DNA. b) In the presence of DNA damage, Asf-1 binds more H3-H4. Rtt109 acetylates H3 on Lysine 56, which weakens H3-Asf-1 interaction. Ubiquitylation of acetylated H3 therefore is a key step in cell allows for dissociation of Asf-1 and H3. c) Rad53 is phosphorylated by Mec1 when there is damage, which turns on the checkpoint. Rad53 auto-phosphorylates. Phosphates PTC2, PTC3, and PPH3 remove the phosphates from Rad53 after cycle recovery. Further we repair. d) CAF-1 is able to bind more (H3-H4)2 to deposit it onto the repaired DNA. Asf-1 dissociates from H3 and de-phosphorylated Rad53 can bind Asf1. The hypothesize that when Asf1 decrease in phosphorylated Rad53 turns off the checkpoint. cannot dissociate from H3-H4 (when H3K56 acetylation by Rtt109 or the subsequent ubiquitylation by Rtt101 are abolished) Asf1 is unable to bind Rad53 and therefore the checkpoint stays activated longer. If the checkpoint persists, CAF-1 will not be able to 16

deposit (H3-H4)2 onto the DNA This pathway is simplified in Figure 1 by demonstrating our proposed scheme. This ultimately would provide more insight into a key mechanism for checkpoint recovery.

Methods and Materials

Strain Design

All strains were transformed via High Efficiency Yeast Transformation (Gietz and

Schiestl, 2007). Strains YLK01 and YLK02 (Table 2) were derived from background strains YMT169 and YMT170 (transformed by Michael Tsabar, Brandeis University,

PhD Candidate), which were derived from YMV80 (Figure 2).

Figure 2: YMV80 derived strains contain one SSA event on ChIII (Clerici et al, 2005) YMT169, YMT170, YLK01, and YLK02 were derived from YMV80 and all contain a single inducible DSB on ChIII. Galactose-induced HO cut site at the Leu2 locus produces an 8kb and a 2.5kb fragment. The repair product is a 3.5kb fragment, which results in a 21.5kb deletion.

Strains YLK03 - YLK15 (Table 2) were derived from background strain, YFA1

(transformed by Fiona Aguilar, senior honors thesis, Brandeis University, 2014), which was derived from YJK17 (Table 1, Figure 3) transformed with linearized pJH592 digested with StuI. PJH592 contained a URA3 HO cut site in a 2.3 kb lambda insert, 17

which repairs via an SSA event (Figure 4). YJK17 was a single DSB model, which made

YFA1 a double DSB model.

Figure 3: YJK17 derived strains contain a GC event (Kim and Haber, 2009) YJK17 contains a galactose-induced HO cut site at MATα on ChIII, which produces a 3.9kb fragment that is detected by a MAT-distal probe. Repair occurs via ectopic gene conversion. ChV contains the donor MATa-inc sequence. Repair via GC results in MAT switching. Vertical blue lines represent EcorI cleavage sites.

Figure 4: YFA1 derived strains contain an SSA event on Chv (Fiona Aguilar, Brandeis University Senior Thesis, 2014) YFA1 was derived from YJK17 by inserting a second galactose-induced HO cut at the URA3 site on ChV, which repairs via single strand annealing. Vertical blue lines indicate BglII cleavage sites. HO produces one 4.5kb and one 3.8kb fragment. The repaired product is a 2.3kb fragment, which resulted in deletion of a 6kb fragment on ChV.

Strain Derivation DSBs YMV80 hmlΔ::ADE mataΔ::hisG hmrΔ::ADE1 leu2-cs ade3::GAL::HO ade1-100 lys5 ura3-52 1 YMT169 YMV80 nat::HPH asf1::TRP 1 YMT170 YMV80 nat::HPH 1 YJK17 MATα hoΔ hmlΔ::ADE1 hmrΔ::ADE1 ade1-100 leu2-3,112 trp::hisG lys5 ura3-52 ade3::GAL::HO 1 arg5,6::MATa-inc::HPH1 YJK113 YJK17 asf1::KAN 1 YJK115 YJK17 asf1::TRP1 cac1::NAT 1 YJK117 YJK17 cac1::NAT 1 YMT129 YJK17 rtt109::KAN 1 YMT130 YJK17 asf1:TRP1 rtt109::KAN 1 YMT131 YJK17 cac1::NAT rtt109::KAN 1 YFA01 YJK17, pJH592 2 YFA02 YFA01 asf1::TRP1 2 YFA03 YFA01 cac1::NAT 2 YFA04 YFA01 asf1::TRP1 cac1::NAT 2 YFA05 YFA01 cac1::NAT rtt109::KAN 2 YFA06 YFA01 rtt109::KAN 2 Table 1: Strains constructed by current and past lab members 18 Strain Derivation DSBs YLK01 YMT170 cac1::NAT 1 YLK02 YMT169 cac1::NAT asf1::TRP1 1 YLK03 YFA1 rtt101::LEU2 2 YLK04 YFA1 asf1::TRP1 rtt101::LEU2 2 YLK05 YFA1 rtt101::LEU2 rtt109::KAN 2 YLK06 YJK17 rtt101::LEU2 1 YLK08 YFA1 asf1::trp1 rtt109::LEU2 2 YLK13 YFA1 hht2 R129E 2 YLK14 YFA1 hht2 R129E rtt101::KAN 2 YLK15 YFA1 hht2 R129E asf1::KAN 2

YLK16 YFA1 hht2 R129E rtt109::KAN 2 Table 2: Strains constructed

Strains were transformed by using antibiotic or amino acid markers (Tables 1 and

2). Transformants were plated on drop-out plates or antibiotic plates to select for

successful transformed colonies. Transformation was confirmed via PCR (Table 3).

Primer Used for CheckNatC Sense primer for cac1::NAT Cac1PPT Antisense primer for cac1::NAT and cac1Δ verification Cac1 ORF Sense primer for cac1Δ verification Leu2 ORF – 25 RP Sense primer for rtt101::LEU2 Rtt101 - 387 Antisense primer for rtt101::LEU2 and rtt101Δ verification Rtt101 +120 Sense primer for rtt101Δ verification FA03 Rtt109 us Sense primer for rtt109::KAN Kan ORF +42 Antisense primer for rtt109::KAN FA5 Rtt109 us ORF Sense primer for rtt109Δ verification FA05 Rtt109 ORF Antisense primer for rtt109Δ verification HHT2 +313 FP Sense primer for hht2 R129E HHT2 -509 RP Antisense primer for htt2 R129E Kan ORF +42 Sense primer for rtt101::KAN Rtt101 - 387 Antisense primer for rtt101::KAN and rtt101Δ verification Rtt101 +120 Sense primer for rtt101Δ verification Asf1us P1 Sense primer for asf1::KAN and asf1Δ verification Kan ORF +42 Antisense primer for asf1::KAN Asf1 RP2-ORF Antisense primer for asf1Δ verification Table 3: Primers

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Viability Assay

Single colonies were inoculated in 5 ml yeast extract peptone dextrose (YEPD) and incubated overnight at 30°C. Cells were washed twice in YEP-lactose (YEP-lac) media to remove galactose repression, then incubated in 5 ml YEP-lac for about 6 hours.

Approximately 100 cells are plated on three YEPD plates and three YEP-galactose (YEP- gal) plates and they are incubated at 30°C for 3-5 days. The strains contain a galactose- induced HO endonuclease DSB. Cells that grow on YEP-gal are termed “survivors” because they repair and recover the DNA damage checkpoint. Percent viability is calculated by dividing number of colonies grown on YEP-gal by number of colonies grown on YEPD.

Time Course

Time courses were performed on the rtt101Δ and rtt109Δ strains with two DSBs by inoculating single colonies in YEPD, incubating overnight, washed with YEP-lactose, and then grown in YEP-lactose. Samples were taken from the growing cultures at certain time intervals after galactose was added. The “0 hour” sample was collected before addition of galactose. DNA was extracted from each time point and was then used for southern blotting.

Southern Blot

Southern blotting was used to quantify repair. DNA was extracted from cells and digested with restriction endonucleases (Table 4) overnight. The digested DNA was precipitated out and run on a 0.8% agarose gel at 100V. The gel was transferred to a 20 membrane and radioactive probes were used to hybridize the necessary DNA fragments

(Table 4). Visualization and quantification was done by a phosphoimager.

Restriction Cleavage Site Probe EcoRI MATα in ChIII, MATa-inc in ChV MATdistal probe BglII URA3 in ChV URA3 probe Table 4: Restriction enzymes and radioactive probes used for southern blots

Results

Strains containing a single SSA event exhibit the same phenotype as strains containing a single GC event. It was previously shown that the YJK17 (Figure 3) strain containing one repairable DSB via ectopic GC has a viability of about 80% (Kim and

Haber, 2009) and YMV80 (one repairable DSB via SSA, Figure 2) has a viability of about 74% (Kim et al, 2011). The viability of YFA1, containing the GC event from

YJK17 and a SSA event (Figure 4), was reported to be 64% (Figure 5) (Fiona Aguilar,

Brandeis University Senior Thesis, 2014). It is important to note that the YMV80 SSA event repairs by about 12 hours and hyperactivates the checkpoint (Vaze et al., 2002) while the smaller SSA event in YFA1 was shown to repair in about 3-5 hours (Fiona

Aguilar, Brandeis University Senior Thesis, 2014). A key result from Aguilar’s project was that asf1Δ was recovery defective in the double DSB system and exhibited the same phenotype as Kim and Haber’s asf1Δcac1Δ with one DSB. Single DSB asf1Δ had a viability of 64% and single DSB cac1Δ had a viability of 63%, while single DSB asf1Δcac1Δ had a viability of about 26%, significantly lower than WT, asf1Δ, and cac1Δ

(Kim and Haber 2009). Double DSB asf1Δ was reported to have a viability of 42% while double DSB cac1Δ had a viability of 62% (Figure 5), making asf1Δ recovery defective in the double DSB model (Fiona Aguilar, Brandeis University Senior Thesis, 2014). 21

GC and GC + SSA Strain Viabilities 80

70

Ectopic GC 60 (1 DSB)

50 * * * Ectopic GC + 40 * SSA (2 DSBs) 30 Percent Viability (%) 20

10

0 WT asf1Δ cac1Δ rtt109Δ asf1Δcac1Δ asf1Δrtt109Δ cac1Δrtt109Δ

Figure 5: Strains constructed and viabilities performed by Fiona Aguilar, Brandeis University Senior Thesis, 2014

To examine if the SSA event in YFA1 derived strains was causing a significant decrease

in viability, YMV80 was mutated with asf1Δ, cac1Δ, or asf1Δcac1Δ. The WT viability

was 74%, which was the same as the reported value from Kim et al, 2011, the asf1Δ was

70%, cac1Δ was 68%, and asf1Δcac1Δ was 52% (Figure 6). Because asf1Δ and cac1Δ in

both single DSB models (GC or SSA) was not significantly different (p = 0.73 and p =

0.75, respectively), and the viability of the double mutant asf1Δcac1Δ in the single SSA

model was significantly lowered when compared to WT (p = 0.003) while it is not

significantly different from the asf1Δcac1Δ GC mutant (p = 0.05), it can be inferred that

adding the SSA event to YJK17 did not cause more of a recovery-defect in the YFA1

derived strains. 22

SSA Viabilities 90 80 70

60 * 50 40 30

Percent Viability (%) 20 10 0 WT cac1Δ asf1Δ asf1Δcac1Δ Figure 6: YMV80 derived strains exhibited similar viability as YJK17 derived strains

Deleting RTT101 caused a recovery defect in both one DSB and two DSB systems.

RTT101 was deleted in YJK17 and YFA1. In the single DSB strain, rtt101Δ had a viability of 47%, compared to WT 68% (Figure 7), which is significantly less (p = 0.01) and therefore making rtt101Δ the only recovery-defective strain with one DSB. The rtt101Δ two DSB strain had a viability of 35%, compared to WT 64%, also being significantly less (p < 0.01), and was therefore also recovery defective.

GC and GC + SSA Strain Viabilities 80

70 Ectopic GC 60 (1 DSB) * 50 Ectopic GC * * * 40 * + SSA (2 DSBs) 30

Percent Viability (%) 20

10

0 WT rtt101Δ asf1Δrtt101Δ asf1Δrtt109Δ rtt101Δrtt109Δ Figure 7: YJK17 and YFA1 derived strain viabilities showing recovery defects in rtt101Δ with both one and two DSBs, asf1Δrtt101Δ two DSBs, asf1Δrtt109Δ two DSBs, and rtt101Δrtt109Δ two DSBs.

23

Asf1, Rtt101, and Rtt109 are epistatic to each other in the two DSB system. It was shown that deleting ASF1 in YFA1 exhibited a low viability (42%) and was therefore recovery defective (Aguilar, 2014). The two DSB rtt101Δ strain was also recovery defective, with a viability of 35% (p = 0.0002 when compared to WT, p = 0.17 when compared to asf1Δ), and the rtt109Δ two DSB strain was also recovery defective (39% viability, p = 0.004 when compared to WT, p = 0.47 when compared to asf1Δ and p =

0.46 when compared to rtt101Δ). Figure 7 displays the viability of double deletions of

ASF1, RTT101, and RTT109 in the two DSB strain. The viability of asf1Δrtt101Δ was

35% (p = 0.02 when compared to WT, p = 0.53 when compared to asf1Δ, p = 0.62 when compared to rtt101Δ), meaning it was also recovery defective, but it did not have a significantly lower viability than the single deleted mutants. This observation suggests that Rtt101 and Asf1 are epistatic. The asf1Δrtt109Δ strain had a viability of 40% (p =

0.003 when compared to WT, p = 0.96 when compared to asf1Δ, p = 0.45 when compared to rtt109Δ) demonstrating that Asf1 and Rtt109 are also epistatic. Lastly, rtt101Δrtt109Δ had a viability of 36% (p = 0.004 when compared to WT, p = 0.80 when compared to rtt101Δ and p = 0.69 when compared to rtt109Δ), also providing evidence for Rtt101 and Rtt109 epistasis. This data led us to hypothesize that Asf1, Rtt101, and

Rtt109 functioned in the same pathway for checkpoint recovery.

Deleting RTT101 or RTT109 does not hinder repair. Once it was determined that deleting RTT101 or RTT109 in the double DSB system led to a decrease in viability and a recovery defect, we sought to determine if rtt101Δ or rtt109Δ strains were able to still repair their DNA. If the mutants impeded repair, that could explain the lowered viability. 24

The ectopic GC event is detected by performing an endonuclease digest on the

DNA using EcoRI. Before HO induction (addition of galactose), 6.5kb and 3kb bands are detected using the MAT-distal probe (Figure 3, Figure 8a,c). After HO induction, the

6.5 kb band is cut to produce a new 3.9kb while the 3kb band (that is produced by the

MATa-inc donor) remains stable (Figure 8a,c). Repair results in the reestablishment of the 6.5 kb band (Figure 8a,c). Interestingly, neither RTT101 or RTT109 deletions resulted in a defect in repair. The rtt101Δ strain showed 93% repair in the GC event by hour 12

(Figure 8a,e) and rtt109Δ showed 71% repair in the GC event by hour 12 (Figure 8c,e).

The cells were able to repair and therefore suggesting that the recovery defect was caused by failure to deactivate the checkpoint.

The SSA event is detected by using the BglII endonuclease to digest the DNA and a URA3 probe. Before HO induction, a 8.3kb band is detected (Figure 4, Figure 8b,d).

After HO induction, a 4.5kb and a 3.8kb band is detected (Figure 4, Figure 8b,d). Once it is repaired, a 2.3kb band is detected (Figure 4, Figure 8b,d). Although it was not quantified, both rtt101Δ and rtt109Δ showed repair.

25

Ectopic GC Repair 100% 90% 80% 70% 60% 50% rtt101Δ 40% rtt109Δ Percent Repair 30% 20% 10% 0% 0 1 3 5 7 9 12 Hour

Figure 8: Southern blots of rtt101Δ and rtt109Δ in the two DSB system and quantified repair

Impairing Asf1-H3 interaction rescues rtt101Δ and rtt109Δ recovery defect when

there are two repairable DSBs. In a previous study (Galvani et al., 2008), it has been

observed that certain point mutations in H3, particularly mutating R129 to a glutamic 26 acid (H3R129E), weakens the interactions between histone-H3 with Asf1. We used this information to test if cell recovery was dependent on Asf1 being dissociated from H3.

The hypothesis was if we observed a higher viability than the original rtt101Δ mutant

(35%), then the R129E mutation “rescues” the rtt101Δ mutant. By weakening the Asf1-

H3 interaction, Rtt101 will not be needed to ubiquitylate H3K56ac and therefore Asf1 will easily bind Rad53, even in the absence of Rtt101. We first mutated YFA1 at the

HHT2 locus (H3) to produce H3R129E mutants. This had a lowered viability of 57%

(Figure 9). We then deleted RTT101 in the R129E mutant. The viability of the H3R129E rtt101Δ mutant was 52% (p = 0.12 when compared to H3R129E and p = 0.01 when compared to rtt101Δ), which is significantly different from rtt101Δ but not significantly different from the H3R129E mutant. This provided evidence that the H3R129E mutation rescued the rtt101Δ mutation.

Histone Mutant Viabilites 70

60

50 * * WT H3 * 40 * H3R129E

30

Percent Viability (%) 20

10

0 WT asf1Δ rtt101Δ rtt109Δ

Figure 9: WT and histone H3 R129E mutants. All strains contain two repairable DSBs.

27

Next we deleted RTT109 in the R129E strain and hypothesized that this should also rescue rtt109Δ because if Asf1 has a low affinity for H3, the cells will not need

Rtt109 to acetylate H3K56, which is originally needed to weaken Asf1-H3 binding. The

R129E rtt109Δ had 54% viability (Figure 9) (p = 0.5 when compared to R129E and p =

0.02 when compared to rtt109Δ). Similar to R129E rtt101Δ, R129E rtt109Δ had a significantly increased viability than the single rtt109Δ mutant and a very similar viability to the R129E mutant. Because the R129E mutation rescued rtt101Δ and rtt109Δ, we hypothesized that weakening the Asf1-H3 interaction was crucial for checkpoint recovery.

We then deleted ASF1 in the H3R129E mutant and the hypothesis was if this had a recovery defect similar to the original asf1Δ, then this would provide more evidence to the proposed pathway because although the H3R129E mutant rescued the rtt101Δ and rtt109Δ, it should not rescue asf1Δ. The cells will lack Asf1 and a recovery defect should be observed, which is exactly what was seen. H3R129E asf1Δ mutants had a viability of

40% (Figure 9) (p = 0.001 when compared to H3R129E and p = 0.51 when compared to asf1Δ). This had a similar viability as the original asf1Δ and was therefore recovery defective, providing evidence that the H3R129E mutation did not significantly lower viability.

Discussion

Asf1 and CAF-1 were said to have overlapping roles in deactivation of the DNA damage checkpoint after repair is completed (Kim and Haber, 2009), but it was then shown that in a two DSB yeast strain, asf1Δ mutants were recovery defective while 28 cac1Δ was not (Fiona Aguilar, Brandeis University Senior Thesis, 2014). This led us to believe that Asf1 had a more significant role in deactivating the checkpoint than CAF-1.

Asf1’s ability to bind Rad53 was a key observation in studying the Asf1 pathway. When

Rad53 is phosphorylated, it continues to signal its downstream targets and the checkpoint persists. But when Rad53 is dephosphorylated and bound to Asf1, it can no longer phosphorylate downstream checkpoint proteins and therefore the cell may re-enter the cell cycle. To observe this, pathways upstream of Asf1 were studied.

Asf1 binds to H3-H4 in the presence of DNA damage (Burgess and Zhang, 2013) and is said to lose binding affinity to H3 when Rtt109 acetylates H3K56 and then further ubiquitylated by Rtt101 (Han, et al., 2013). We proposed the hypothesis that the acetylation and ubiquitylation of H3K56 by Rtt109 and Rtt101, respectively, is a key process for the Asf1 hand-off from H3 to Rad53, and therefore a crucial process in checkpoint deactivation.

Our first analysis was to observe if Asf1, Rtt101, and Rtt109 were epistatic to each other in the two DSB system. The mutants were not epistatic to each other in the single break system, because asf1Δ and rtt109Δ were not recovery defective while rtt101Δ was, leading us to believe that it is involved in another pathway during DNA damage recovery. While the single asf1Δ, rtt101Δ, and rtt109Δ mutants all exhibited low viabilities, the double mutants asf1Δrtt101Δ, asf1Δrtt109Δ, and rtt101Δrtt109Δ did not have significantly lowered viabilities, providing evidence that these genes behave epistatically to each other and function in the same pathway.

There is, however, some contradictory data that must be addressed. Strains containing a single repairable DSB and single deletions of ASF1 or RTT109 were not 29 recovery-defective, while deletion of RTT101 showed a recovery defect, which therefore displays incomplete epistasis between these genes. We hypothesize that Rtt101 could be a link between the Asf1 and CAF-1 pathway because they have overlapping roles in checkpoint recovery and CAF-1 binding H3-H4 is a key step in histone deposition after repair. Also, the cac1Δrtt109Δ mutant in the single DSB strain did not have a recovery defect. This was contradictory to our data because if RTT109 is truly epistatic to ASF1, then the cac1Δrtt109Δ single DSB mutant would have behaved similarly to the asf1Δcac1Δ mutant, which was recovery-defective. One possibility for this is that Asf1 is needed more than CAF-1 to bind Rad53 and aid in deactivating the checkpoint, but when cells lack both of them, they will have very limited ability to regulate the checkpoint. The cac1Δrtt109Δ will still have Asf1 and because it is a single break, we hypothesize that

Rad53 expression and hyperphosphorylation will not overwhelm Asf1. Current work in the lab is focusing on Rad53 overexpression and hyperphosphorylation in the single and double DSB models. The hypothesis is that Rad53 is overexpressed and hyperphosphorylated to a greater extent in the two DSB strain than the single DSB strain.

After supporting the hypothesis that Asf1, Rtt101, and Rtt109 function in the same pathway for checkpoint recovery, we wanted to make sure that the mutants were still able to repair the DNA lesions and that the recovery defect was truly an issue with the proper machinery to deactivate the checkpoint. To observe this, southern blot analysis was performed to visualize and quantify repair. Previous data (Aguilar, Brandeis

University Senior Thesis, 2014) revealed that asf1Δ in the double DSB model could repair and that the recovery defect was caused by an inability to deactivate the checkpoint. In this project, rtt101Δ and rtt109Δ in the two DSB strain were both able to 30 repair normally. This led us to believe that deleting these three genes did not impair cellular repair mechanisms, but impaired cellular ability to re-enter the cell cycle after

DNA damage-induced arrest.

Next we wanted to determine what the consequences of weakening the Asf1-H3 interaction would be for checkpoint recovery. This was done because if Asf1 dissociation depends on Rtt109 and Rtt101 acetylation and ubiquitylation on H3K56, respectively, then already having a dissociated Asf1 from H3 in rtt101Δ or rtt109Δ mutants will rescue those cells and allow for Asf1 to bind to Rad53 and subsequently aiding in the deactivation of the checkpoint. Mutating H3 at R129 to a glutamic acid was reported to weaken Asf1-H3 interactions (Galvani et al., 2008). Mutations in Asf1 had been reported to also weaken the Asf1-H3 interactions (Jiao et al., 2012), but since H3 and Rad53 bind to Asf1 at the same site (Jiao et al., 2012), mutating Asf1 would not have been a plausible experiment, since we were especially interested in Asf1-Rad53 binding. H3R129E rtt101Δ and H3R129E rtt109Δ mutants with two repairable DSBs were seen to have significantly higher viability than the single rtt101Δ and rtt109Δ mutants, providing evidence that in the absence of Rtt101 or Rtt109, the cells could still recover from the checkpoint because Asf1 was already dissociating from H3.

There are still unanswered questions about the mechanisms involved in deactivating the DNA damage-induced checkpoint. For example, it was unknown why the cac1Δrtt109Δ single DSB mutant had a wild type-like viability while the asf1Δcac1Δ single DSB mutant was recovery defective. If Asf1 and Rtt109 are truly epistatic, then cac1Δrtt109Δ should have behaved like asf1Δcac1Δ, which it did not, suggesting Asf1 and Rtt109 are not completely epistatic. Also, rtt101Δ was the only mutant that was 31 recovery defective in the single DSB model, while asf1Δ and rtt109Δ did not suffer a significant recovery defect. This also suggests that Rtt101 is not completely epistatic to

Asf1 and Rtt109.

Future projects that could be performed would be constructing single and double

DSB strains containing a triple asf1Δcac1Δrtt101Δ mutant to observe if the cells had a lowered recovery defect than the asf1Δcac1Δ or asf1Δrtt101Δ mutants. Another project that is currently being worked on is to co-immunoprecipitate (Co-IP) Asf1-Rad53 and

Asf1-H3 from cells that repair damage but are recovery defective, like the two DSB asf1Δ or rtt101Δ mutants. If Asf1 is bound to H3 and not Rad53, this would provide some evidence that the recovery defect is being caused by Asf1 failure to dissociate from

H3 and bind Rad53.

Another pending question that could be addressed is if rtt101Δ or rtt109Δ are adaptation defective. In a previous study (Kim and Haber 2009), it was published that asf1Δ, cac1Δ and asf1Δcac1Δ mutants were not adaptation defective. In other words, when the cells lacked Asf1 and CAF-1 were not responsible for cells re-entering the cell cycle via adaptation (recovery in the absence of repair) (Kim and Haber 2009).

Determining if rtt101Δ or rtt109Δ mutants are adaptation defective would be an interesting addition to the information about the recovery pathway.

32

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