So Much to Do, So Little Time… Unraveling the Molecular Mechanisms of DNA Helicases 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 Mutation 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 helicase 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 RECQ1 siRNA L1 100 RECQ1-/- 100 RECQ1 siRNA L2 75 75 50 50 C L1 L2 Total DNA content (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 nucleases. 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 DNA2 Pol 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 Homologous recombination 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 nuclease 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 ( 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 (%) 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 mutations 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.