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Homologous Recombination & Double-Strand Break Repair: Crossing-Over with Cancer Biology

Homologous Recombination & Double-Strand Break Repair: Crossing-Over with Cancer Biology Scott Morrical Dept. of Biochemistry smorrica@zoo.uvm.edu Lessons from Prokaryotes & Yeast 2. DSBR in Humans-- Mediators, Paralogs, & BRCA1/2. Types of Recombination:

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Homologous Recombination & Double-Strand Break Repair: Crossing-Over with Cancer Biology

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  1. Homologous Recombination & Double-Strand Break Repair: Crossing-Over with Cancer Biology Scott Morrical Dept. of Biochemistry smorrica@zoo.uvm.edu Lessons from Prokaryotes & Yeast 2. DSBR in Humans-- Mediators, Paralogs, & BRCA1/2

  2. Types of Recombination: • Site-specific recombination. Recombination occurs at defined, short sequences in DNA. Requires a site-specific recombinase enzyme that recognizes the target sequence. • Non-homologous or illegitimate recombination. Little or no sequence specificity or homology requirement. Certain types of transposition; non-homologous end joining. • General or homologous recombination. Occurs between any homologous DNA sequences of sufficient length. Meiotic crossing-over; DNA repair.

  3. Homologous Recombination & Cancer: Why You Should Care Homologous recombination is required for accurate repair of DNA double-strand breaks (DSBs). Therefore protective against carcinogenesis. Errors --> increased mutation rates & susceptibility to carcinogenesis. Nijmegen chromosome breakage syndrome (NBS) Ataxia telangiectasia (AT) AT-like disorder (ATLD) Bloom’s syndrome (BLM) Fanconi's anemia (FA) Werner’s syndrome (WRN) Functions of human breast/ovarian cancer susceptibility genes BRCA1 & BRCA2 are clearly linked to homologous recombination and double-strand break repair. Aberrant recombination phenotypes associated with neoplastic states-- i.e. hyper-recombination in p53 mutants.

  4. Homologous recombination & DSBR are mechanisms of tumor cell resistance to radiation and chemotherapy. Targeting recombination pathways in tumor cells could increase efficacy. 5. Targeted homologous recombination is desirable for cancer gene therapy approaches, i.e. introduction of suicide genes at benign locations in the genome.

  5. Holliday Model of Homologous Genetic Recombination

  6. Mitotic Recombination: Double-Strand Break Repair Model ZAP!! Broken Chromosome Nucleolytic Processing 3’ 3’ DNA Strand Exchange (HR) 3’ 3’ Undamaged Homologous Chromosome DNA Synthesis (RDR) Endonucleolytic Resolution & Ligation Repaired Chromosome

  7. Recombination Lessons from Prokaryotes: The E. coli RecA Paradigm

  8. Structure, Function & Evolution of DNA Repair Enzymes Phylogenetic Diversity of RecA Family RadA Pf hDMC1 Yp2 hRAD51 XRCC3 XRCC2 Uu hRAD51B Ll2 RB69 hRAD51D Pf Dr hRAD51C RadB T4 Ec UvsX RecA Os

  9. ATP ATP ATP ATP ADP ADP ADP ADP Types of DNA Rearrangements Catalyzed by E. coli RecA 2-strand reannealing: + 3-strand exchanges: + + + 4-strand exchanges: + +

  10. Properties of E. coli RecA Protein • Protomeric m.w. = 38 kDa. • Binds cooperatively to ssDNA at neutral pH; complex • stabilized by (d)ATP or ATPgS, destabilized by ADP. • dsDNA binding requires low pH, ATPgS, or transfer or • nucleation from ssDNA. • Forms filaments on & off of DNA. • Presynaptic filament-- RecA filament assembled on ssDNA in • presence of Mg(d)ATP-- is catalytically active form. • Catalyzes DNA-dependent (d)ATP hydrolysis. • Catalyzes (d)ATP-dependent DNA rearrangements including • complementary strand reannealing & homologous 3- or • 4-strand strand exchanges. • Co-protease: In response to DNA damage, facilitates auto- • proteolytic cleavage of LexA repressor which induces the • SOS response in E. coli.

  11. Electron Micrograph of Relaxed Circular dsDNA Molecule Coated with RecA Protein in Presence of ATPgS • Open, right-handed helical filament • DNA is markedly extended and underwound

  12. Story et al.: X-ray Crystallographic Structure of E. coli RecA-ADP Complex (Single Subunit Shown) • RecA crystallizes as helical polymer even w/o DNA • DNA binding loops L1 & L2 are disordered Presynaptic Filaments

  13. The RecA Paradigm of Homologous Strand Transfer Presynapsis RecA Homologous dsDNA ssDNA ATP, SSB 3’ Synapsis 5’ + ATP ADP ATP ADP Branch Migration

  14. Other Recombination Proteins Affect DNA Strand Exchange • Nucleases/helicases generate ssDNA substrates for • presynapsis. • ssDNA-binding proteins (SSBs)-- promote presynapsis*, • sequester displaced strand in branch migration. • Recombination mediator proteins (RMPs)-- assemble • presynaptic filament. • DNA helicases/translocases-- promote branch migration, • filament remodeling.

  15. Problems in Presynaptic Filament Assembly: • Targeting filament assembly onto ssDNA in the presence of • excess cellular dsDNA. • Competition between RecAs and abundant cellular SSBs for • binding to ssDNA. • Order of Addition Effect: • -- SSB added to ssDNA after preincubation of RecA + ssDNA + ATP • gives optimal stimulation of filament assembly, ATPase, & strand • exchange activities. • -- SSB preincubated with ssDNA before RecA + ATP added gives • strong inhibition of filament assembly, ATPase, & strand exchange. • Both problems dealt with by Recombination • Mediator Proteins (RMPs) & other factors

  16. Evolutionary Conservation of Recombinase, SSB, & Mediator Functionalities Gp32 T4 UvsX-ssDNA Presynaptic Filaments UvsX T4 phage E. coliS. cerevisiaeH. sapiens Recombinase: UvsX RecA Rad51 Rad51 SSB: Gp32 SSB RP-A RP-A Mediator(s): UvsY RecO/R Rad52 Rad52 RecF? Rad55/57 Rad51B,C,D? Xrcc2,3? Brca2?

  17. Enzymology of DSBR • Yeast RAD52 Epistasis Group • Human Rad51 paralogs • The BRCA connection

  18. How (Unprogrammed) DNA Double-Strand Breaks Occur • Ionizing Radiation (i.e. X- & g-rays) and some chemical agents locally disrupt the backbones of both strands of B-form DNA. 2. Inappropriate cleavage of dsDNA by an endonuclease. 3. BER or NER enzyme processing of interstrand crosslinks or of base lesions too close to nicks on the opposite strand. • Replication fork collapse: --Replication past a single-strand disruption or nick. --Replication fork collisions with cleavage complexes of type I & II topoisomerases. 5.Deoxyribonucleotide starvation.

  19. Implications for Cancer Treatment • Radiation: Hope that rapidly proliferating tumor cells won’t be • able to repair induced DSBs fast enough, & therefore selectively • undergo apoptosis. Problems-- resistant cells are good at DSBR; • doesn’t work well for slower-growing tumors; secondary effects. • Topoisomerase poisons: Stabilize topo-DNA cleavage complexes, • increase frequency of replication fork collapse in rapidly proliferating • tumor cells. Topo-I + camptothecin nick + + ? Topo-II + m-AMSA +

  20. Hydroxyurea: Inhibitor of ribonucleotide reductase. • Chemotherapy deprives rapidly proliferating tumor cells of • deoxyribonucleotide precursors for DNA synthesis & repair. • Observation: DSBs accumulate in treated cells- why? • Many stalled replication forks; get converted into • mitotic DSBs (?) • Inability to complete replicative steps of DSBR • pathways.

  21. RAD52 Epistasis Group In Yeast (& Humans) Genetically Implicated in Homologous Recombination & Double-Strand Break Repair

  22. Mitotic Recombination: Double-Strand Break Repair Model ZAP!! Broken Chromosome Nucleolytic Processing 3’ 3’ DNA Strand Exchange (HR) 3’ 3’ Undamaged Homologous Chromosome DNA Synthesis (RDR) Endonucleolytic Resolution & Ligation Repaired Chromosome

  23. RAD52 Epistasis Group: Genes & Gene Products (All conserved in humans in one way or another) Processing: MRE11 Mre11/Rad50/Xrs2 complex (MRX) RAD50 implicated in nucleolytic resection of XRS2 (NBS1)DSBs --> 3’ ssDNA tails Recombination: RAD51 Ortholog of E. coli RecA RAD52 Mediator, annealing & strand exchange protein RAD54 Snf2/Swi2 ATPase RAD55 Rad51 paralogs;Rad55/Rad57 dimer = mediator RAD57 (Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3) RAD59 Rad52 paralog RDH54 Rad54 paralog RFA1 Lg. Subunit of RPA (SSB) heterotrimer

  24. Mediator Rings & Oligomers “7-11” Rad52 Single-strand Annealing Strand Exchange

  25. Mediator Function Of Yeast Rad52 Yeast Rad52 relieves RPA order of addition effect in Rad51-catalyzed DNA strand exchange assay… Rad51 -> RPA RPA+Rad52 -> Rad51 RPA -> Rad51 72 min …but Rad52 does not replace RPA in strand exchange; rxns remain RPA-dependent. 36 min

  26. Biochemical Demonstration of Yeast Rad51-Rad52 Interactions Immunoprecipitations: From wt extracts Affinity Chromatography: From Rad52 overexpresser Sung et al.

  27. N-terminal Fragment of Human Rad52 (Residues 1-209) Promotes Reannealing & Crystallizes as an Undecameric Ring bbba I II Singleton et al.; Kagawa et al.

  28. Propagation of Putative ssDNA Binding Site Around the Ring Surface of HsRad521-209

  29. Yeast Rad54 • Member of Snf2/Swi2 family of DNA-dependent • ATPases/motor proteins/helicases. • Binds to dsDNA and introduces local and global changes • in superhelicity consistent with translocation along duplex • without unwinding. • Binds to Rad51 and stimulates DNA strand exchange rxns. • Overcomes dsDNA inhibition of Rad51-catalyzed DNA • strand exchange. • Rad51 differs from E. coli RecA in having an • intrinsically high affinity for dsDNA-- the dsDNA • can actually sequester Rad51 & thereby inhibit • strand exchange initiated from ssDNA.

  30. Heyer & co-workers: Rad54 Disassembles Inappropriate Rad51- dsDNA Complexes & Thereby Facilitates Appropriate ssDNA- Initiated DNA Strand Exchanges Rad54 may also promote nucleosome rearrangements around target sequence in homologous duplex.

  31. Mre11/Rad50/Xrs2 (MRX) Complex (Yeast & Human Versions) • Localizes with nuclear “repair foci” following cell • exposure to ionizing radiation. • Implicated in resection of DSBs into 3’ ssDNA tails • -- curious, since Mre11 is a weak 3’ --> 5’ exonuclease! • -- Mre11 also has ssDNA endonuclease activity • -- all Mre11 nuclease activities Mn++ dependent • Mre11 & Rad50 are conserved in all kingdoms of life. • Xrs2 is only weakly conserved. Human Mre11/Rad50 • complex associates with Nbs1, which is deficient in NBS, a • rare cancer-prone syndrome. Hypomorphic alleles of Mre11 • cause A-TLD, a human chromosomal instability syndrome.

  32. Proposed Role of MRX in DSB Resection in Yeast Wild-type MRX: weak unwinding activity or recruits helicase. mre11-H125N: still unwinds but lacks ssDNA endonuclease.

  33. Electron Microscopy Of Yeast Rad50-Mre11 Complexes Rad50 + Mre11 (2:2) (2:1) Mre11 Anderson et al., J. Biol. Chem., Vol. 276, Issue 40, 37027-37033, October 5, 2001

  34. Rad50:Member of SMC Family. Walker A & B ATP-Binding Motifs Separated by Long Coiled-Coil Domain, Used (?) to Orient Mre11 Subunits & Link DSB Sites

  35. Human (Vertebrate) Rad51 Paralogs: Rad51B Rad51C Rad51D Xrcc2 Xrcc3 Implicated in Homologous Recombination & Repair; Formation of Nuclear Rad51 Foci Following IR Exposure, Etc.

  36. Rad51B Knockouts in Chicken B Lymphocyte DT40 Cells Compromise Rad51 Repair Foci Induced by DNA Damaging Agents Takata et al.

  37. Human Rad51 Paralogs Form Two Distinct Complexes: (West, Sung, & other labs) BCDX2 CX3

  38. BCDX2-- 1:1:1:1 Stoichiometry CX3-- 1:1 Stoichiometry

  39. Summary: Biochemical Activities Ascribed to Rad51 Paralog Assemblies & Sub-Assemblies ss-Binding X (+ HJ, duplex) X X X X (+ duplex) X (+ nicks) X ATPase X X X X X Mediator* X(Rad51 ATP/ADP exchange) X Strand Ex* X X (no ATP?) X (no ATP?) X (no ATP?) Complex B C D X2 BC DX2 BCDX2 X3 CX3 *Caution necessary since C and CX3 have weak strand exchange activities.

  40. Role of Breast/Ovarian Cancer Susceptibility Genes BRCA1 & BRCA2 In Homologous Recombinatin & DNA Repair

  41. Nobody Said It Would Be Simple…

  42. … But Evidence Suggests Brca2 Plays a Direct Role and Brca1 an Indirect Role in Promoting Rad51-Dependent Recombinational Repair

  43. Brca1 Knockout Reduces Efficiency of Rad51 Repair Foci Following Cisplatin or IR Exposure of Mouse ES Cells Bishop & co-workers

  44. IR-Induced Rad51 Foci Formation Requires Brca2 (Spontaneous Rad51 Foci That Occur During S-Phase Are Brca2-Independent) Cells contain Brca2 mutant lacking nuclear localization signal; Brca2 stays in cytoplasm. West

  45. X-ray Structure of Mouse/Rat Brca2 ssDNA-Binding Domain Complexed to Dss1 & ssDNA Yang et al. (2002) Science 297, 1837-1848

  46. Structure of Mouse Brca2 DNA-Binding Domain: D = Intact DBD- Dss1 complex E = Tower deletion DBD mutant bound to Dss1 & oligo dT9 OB-fold:Oligonucleotide/ oligosaccharide binding fold, structurally conserved.

  47. Brca2DBDDTower-Dss1-dT9 Complex at 3.5 Å 5 of 9 ssDNA Residues Resolve, Bound Across OB2 & OB3

  48. X-ray Structure of Human Rad51 RecA Homology Domain Complexed to Brca2 BRC Repeat Pellegrini et al. (2002) Nature 420, 287-293

  49. 1.7 Å Structure of Human BRCA Repeat 4 (A.A. 1517-1551) Bound to RecA Homology Domain of Rad51 (S95 - C-Terminus) An Ingenious Trick: BRC4 fused to N-terminus of truncated Rad51 via flexible linker-- suppresses natural tendency of Rad51 to self-aggregate! Rad51 Rad51 BRC4 BRC4 HsRad51 vs. EcRecA

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