So Much to Do, So Little Time… Unraveling the Molecular Mechanisms of DNA Associated with Human Disease and Aging

Robert M. Brosh Jr. Section on DNA Helicases Laboratory of Molecular Gerontology National Institute on Aging, NIH DNA DAMAGE AND REPAIR

Intrinsic: Chemical agents: Radiations: Spontaneous base Endogenous Exogenous Ultraviolet Ionizing modification, (oxygen (chemical radiation radiation Replication errors radicals) mutagens)

Gox U

AP Site

BASE EXCISION NUCLEOTIDE RECOMBINATIONAL REPLICATION REPAIR EXCISION REPAIR FORK REPAIR RESTORATION

Genomic instability Cell survival Cell death Chromosomal aberrations

Disease Aging Cancer LABORATORY OF MOLECULAR GERONTOLOGY

Section on DNA Section on Base Helicases Excision Repair Section on DNA Repair

Section on Antibody Diversity Unit on Telomeres Section on Gene Targeting NIH Biomedical Research Center Question: Why are there unique clinical and cellular phenotypes in the RecQ disorders? HELICASE RQC HRDC RECQ1, H. sapiens 649 aa BLM, H. sapiens 1417aa EXO WRN, H. sapiens 1432 aa

RECQ4, H. sapiens 1208 aa RECQ5, H. sapiens 991 aa

Objective: To delineate and characterize unique roles of RecQ helicases in genomic stability maintenance

Today: 1. RECQ1 cellular phenotypes 2. Model system to study WRN genetics 3. Novel functions of FANCJ helicase Roles for RecQ Helicases in DNA Metabolism “Damage” encountered during replication of the genome

1. Road Block Remover Resolution of Tetraplex - Resolve alternate DNA Secondary structures for proper Structure Resolution replication progression

Fork Regression

WRN Proofreading DNA Damage Sensing Branch Fork Migration 2. Quality Control - Restore replication fork progression - Complete proper repair - Prevent inappropriate recombination HELICASE RQC HRDC NLS

RECQ1, H. sapiens 649 aa

• First RecQ helicase discovered in the early 1970’s by Blackshear and Okumura Labs • Smallest RecQ helicase • Most abundant human RecQ helicase • Not genetically linked to a human disease • No organismal phenotypes for RECQ1 KO mouse detected by Blackshear Lab, NIEHS Biochemical Activities of Recombinant Human RECQ1

Helicase Activity

RECQ1 (nM) -

Substrate - Unwinds synthetic replication forks and Holliday Junctions - Melts D-loop recombination Product intermediate

Strand Annealing Activity

Time (min) 0 0.5 1 2 4 8 16 32 fork

Product - Modulated by ATP binding due to conformational change in RECQ1

Substrate

Sharma et al., J. Biol.Chem., 2005 What DNA metabolic pathways does RECQ1 participate in?

• Identify RECQ1 interacting partners and characterize their functional interactions

RECQ1 helicase interacts with DNA repair factors that regulate genetic recombination

RECQ1 RECQ1 Rad51 MSH6 RPA14 RPA70 MSH2 EXO1 RECQ1-Rad51 RPA30 found in MutS / MLH1 RECQ1 MLH1-PMS2 complex RPA stimulates Sharma et al., PLOS One 2007 RECQ1 helicase Cui et al., Nucl. Acid Res. 2004 PMS2 - RECQ1 stimulates EXO-1 incision - MSH2/6 stimulates RECQ1 helicase Doherty et al., J.Biol.Chem. 2005 RECQ1 Suppresses Sister Chromatid Exchanges Wild type RECQ1-/-

Cell line metaphases No. of scored SCE/metaphase Wild type MEF 20 2.71 ± 0.932 RECQ1 knockout MEF 25 12.32 ± 1.416 Sudha Sharma

Sharma et al., MCB, 2007 Spontaneously elevated H2AX and Rad51 foci in RECQ1 knockout MEFs Reduced cell growth and elevated sister chromatid exchange in RECQ1-depleted cells

Colony forming assay

Control shRNA RECQ1 shRNA

BrdU labeled metaphase chromosome spreads

Sharma et al., PLOS One, 2007 Control siRNA RECQ1 siRNA Cellular Deficiency of RECQ1 Leads to Increased IR Sensitivity

primary MEFs of RECQ1 knockout siRNA knockdown of RECQ1 mice are sensitive to IR leads to increased IR sensitivity in HeLa cells

125 125 RECQ1+/+ control siRNA 100 RECQ1 siRNA L1 RECQ1-/- 100 RECQ1 siRNA L2 75 75

50 50 C L1 L2 Total DNA contentTotal (as of % untreated) Total DNA content (as of % untreated) RECQ1 25 25 Actin 0 0 0246810 0246810 IR (Gy) IR (Gy) Does RECQ1 respond to DNA damage? Untreated + IR

RECQ1 is a nucleolar protein and relocates to form chromatin bound foci upon DNA damage

Untreated IR (10Gy) Endogenous RECQ1 associates P1 S2 P2 S3 P3 S4 P4 P1 S2 P2 S3 P3 S4 P4 with chromatin in response to IR

RECQ1

Lamin B

Histone H4

Who phosphorylates RECQ1, λ phosphatase-1 - - + + and is this important? IR (10Gy) - + - +

PhosphoRECQ1 RECQ1 is phosphorylated RECQ1 in response to IR Ig Heavy chain

DNA Damage

Homologous Recombination

+ RECQ1 -RECQ1

Error-free DNA repair Unresolved recombination events Replication restart Elevated sister chromatid exchange

Stable genome Chromosomal instability

Carcinogenesis?

RECQ1 preserves genomic integrity through its role in homologous recombinational repair We are interested in the importance of protein interactions between RecQ helicases and structure specific .

Human Rad2 Structure-Specific Nucleases FEN-1 N I 380 EXO-1 N I 846 XPG N I 1146 WRN and BLM helicases interact with human FEN-1 and stimulate FEN-1 nucleolytic activities.

WRN and RECQ1 helicases interact with human EXO-1 and stimulate EXO-1 nucleolytic activities.

How are these interactions important in vivo? Perhaps under conditions of replicational stress. Hypothesis: WRN stimulates FEN-1 cleavage in vivo to rescue the DNA replication and repair phenotypes of the dna2 replication mutant

Okazaki fragment processing model RPA

Pol  DNA2 FEN-1

PCNA RFC

• FEN-1 over-expression rescues dna2 mutant phenotypes

• Functionally conserved roles of human and yeast FEN-1 in DNA replication and repair WRN Rescues Replication Defects of dna2

dna2-1/WRN940-1432 dna2-1/WRN940-1432

dna2-1/RAD27 dna2-1/RAD27 dna2-1/WRN dna2-1/WRN

DNA2/vector DNA2/vector dna2-1/vector dna2-1/vector 23ºC 37ºC

• Complementation regulated by level of WRN expression • FEN-1 interaction domain of WRN sufficient for rescue • WRN rescues cell cycle progression defect • WRN rescues sensitivity to replication inhibitor HU or DNA damaging agent MMS Sharma et al., 2004 Human Mol. Genet. Does the WRN: EXO1 interaction play a role in the replication stress response? We chose to use yeast as a model system to answer this question since it was previously shown that yeast and human EXO1 are functional homologs. ACIDIC EXO REPEATS HELICASE RQC HRDC NLS WRN

C-WRN940-1432

WRN expression construct Monika Aggarwal Yeast rad50 mutant strain

Xrs2 Complex (Rad50-Mre11-Xrs2) Pleiotropic effects DNA repair deficiency Hyper-recombination  Nonhomologous end-joining Telomere shortening  Intra-S phase Checkpoint defect DNA damage sensitivity

Overexpression of Exo1 (5’-3’ exonuclease) rescues MMS and IR sensitive phenotypes of Rad50-Mre11-Xrs2 complex mutants, and this is dependent on EXO-1 activity.

Tsubouchi, H and Ogawa, H. Mol. Biol. Cell 2000; 11:2221–33. Moreau, S. et al., Genetics 2001; 159:1423–33. Lewis, KE et al., Genetics. 2002;160(1):49- 62. Lewis KE et al., Genetics 2004; 166(4):1701-13. Test WRN for rad50 rescue ACIDIC EXO REPEATS HELICASE RQC HRDC NLS WRN

C-WRN940-1432

1000 1000

100 RAD50 + Vector RAD50 EXO1 + Vector

100 ) ) % % 10 rad50 + WRN

Survival ( rad50 exo1 + Vector Survival ( Survival 10 1

rad50 exo1 + WRN rad50 + Vector 0.1 rad50 + C-WRN 1 0 0.1 0.2 0.3 0.4 0 0.1 0.2 0.3 MMS (mM) MMS (mM) Expression of full-length WRN WRN rescue is EXO1-dependent rescues rad50 MMS sensitivity Test WRN catalytic domain mutants for rad50 rescue ACIDIC EXO REPEATS HELICASE RQC HRDC NLS WRN E84A K577M

1000 WRN helicase, but not exonuclease activity, is required for genetic 100 rescue of rad50 MMS sensitivity.

10 rad50 + WRN rad50 + WRN E84A Survival (%) Survival

1 rad50 + Vector

rad50 + WRN K577M

0.1 0 0.1 0.2 0.3 0.4 MMS (mM) Can WRN prevent mitotic catastrophe in rad50 mutant?

FACS Analysis No MMS 0.3 mM MMS

RAD50 + Vector

rad50 + Vector

rad50 + WRN

G1 G2 G1 G2

WRN expression prevents accumulation of sub-G1 Can WRN expression rescue IR sensitivity of rad50 ?

1000

100 RAD50 + Vector

Survival (%) rad50 + WRN 10 rad50 + Vector

1 0 20 40 60 80 100 Dose (Gy) WRN expression does not rescue IR sensitivity of rad50, suggesting that WRN rescue is specific to agents like MMS that stall replication forks. Can WRN stimulate EXO1 to process a stalled replication fork to counteract fork reversal?

5’ Mol Cell. 2005 Jan 7;17(1):153-9.

EXO1 (0.25 nM) - + + + + + - - - - Exo1 processes stalled replication WRN (nM) - - 1 2 4 8 1 2 4 8 forks and counteracts fork reversal in checkpoint-defective cells. Substrate

Cotta-Ramusino C, Fachinetti D, Lucca C, Doksani Y, Lopes M, Sogo J, Foiani, M.

1 nt

WRN stimulates the exonuclease activity of EXO1 on replication fork lagging strand, suggesting mechanism to prevent fork regression. Summary

- WRN rescues rad50 MMS sensitivity in EXO-1 dependent manner

- WRN does not rescue rad50 IR sensitivity, suggesting WRN:EXO1 interaction is important for response to agents that induce replicational stress, not direct strand breaks

- WRN rescue of rad50 MMS sensitivity requires helicase, but not exonuclease activity

- WRN prevents MMS-induced mitotic catastrophe in rad50 mutant

- WRN stimulates EXO1 to process replication fork structures in a manner that would counteract fork reversal Understanding the Consequences of Helicase Dysfunction for Age-related Disease, Cancer, and Genomic Instability

SF2 Helicase Disease / Abnormality

WRN Werner syndrome

BLM Bloom syndrome

RECQ4 Rothmund-Thomson syndrome

RECQ1 ?

RECQ5 ?

FANCJ Fanconi anemia, Breast cancer

Common pathways promote chromosomal rearrangements in different genome instability syndromes Distribution of FANCJ and breast cancer associated sequence changes in FANCJ protein

MLH1 binding domain R798X P47A Q944E

1 39 57 245 258 385 398 610 624 689 710 748 775 819 836 888 1063 1249

I IaIa IIII III IV V VI VI

128 158 BRCA1-binding domain Q255H H396D W647C R707C R798X R251C

Fe-S domain 270-363 M299I

-HTCVHPEVVGNFNRNEKCMELLDGKNGKSCYFYHGVHKISDQHTLQTFQGMCKAWDIEELVSLGKKLKACPY-

A349P FANCJ Helicase Family BRCA1 human ATPase/Helicase Domain Binding Domain FANCJ FA, breast cancer XPD Xeroderma pigmentosum, NER defect ChlR1 sister chromatid cohesion defect

yeast Chl1 sister chromatid cohesion defect RAD3 NER defect

mouse RTEL telomere instability ChlR1 sister chromatid cohesion defect

C. elegans DOG-1 instability of guanine-rich tracts Does FANCJ Unwind G4 DNA?

Yuliang Wu S  TP-G4 100 M NE WT-ATP WT+ATP WT+ADP WT+ATP NE WT-ATP WT+ATP K52R-ATP K52R+ATP M 80

60 TP-G4 Forked duplex (19 bp)

40 Unwound (%)

20

0 ssDNA 0 1 2 3 4 5 FANCJ (nM)

G4 unwinding by FANCJ is dependent on Wu et al., MCB 2008 ATP hydrolysis and protein concentration. O S

N N O

 O Effect of telomestatin (5 M) N N exposure on FANCJ-depleted cells N O N

O O N N

O O

20 Telomestatin HeLa control siRNA 2

15 ) 5 ) 1.5 450 HeLa FANCJ siRNA -OD 10 1 370

U2OS control siRNA (OD Cell Number (10 Cell Number 5 BrdU Incorporation 0.5

U2OS FANCJ siRNA 0 0 Hela HeLa U2OS U2OS 0 1 2 3 4 5 control FANCJ control FANCJ Day siRNA siRNA siRNA siRNA

FANCJ depletion sensitizes cells to telomestatin Telomestatin induces elevated γ-H2AX foci in FANCJ-depleted cells -H2AX Merge DAPI

control siRNA No treatment

FANCJ siRNA

control siRNA 5 m TMS 2 hr treatment, 2 hr recovery FANCJ siRNA Proposed Role of FANCJ to Resolve G4 Quadruplex DNA Structures During Replication 3’

(GGG)n

5’

3’

5’

- FANCJ + FANCJ

Genomic Instability 3’ 3’

5’ 5’ Role of FANCJ to Preserve Chromosomal Integrity • Promote S phase progression by resolving DNA roadblocks such as G4 tetraplexes that destabilize or impede the replication fork • Preserve genomic stability to prevent cancer susceptibility FANCJ & Human RecQ Helicases— Potential Cancer Therapy Targets?

Exploit the roles of DNA helicases in DNA repair to enhance cytotoxic effects of DNA damaging agents

Gupta and Brosh, Curr. Med. Chem. 2007 Josh Sommers

Yuliang Wu

Suhasini LMG, NIA-NIH Naga Vilhelm Bohr Michael Seidman Monika David Wilson Yie Liu Aggarwal Pat Gearhart

Perry Blackshear, NIEHS Debbie Stumpo, NIEHS Section on DNA Helicases Ian Hickson, Univ. Oxford Laboratory of Molecular Gerontology Sharon Cantor, U Mass Med Ctr NIA-NIH Alex Mazin, Drexel Univ.