Homologous Recombination: Presynaptic Filaments & Brca2 Scott Morrical Dept. of Biochemistry

1. Homologous Recombination: Presynaptic Filaments & Brca2 Scott Morrical Dept. of Biochemistry smorrica@zoo.uvm.edu E. coli RecA Paradigm Eukaryotic Rad51 Rad51-Brca2 Interactions

2. Literature 1: Bianco PR, Tracy RB, Kowalczykowski SC. DNA strand exchange proteins: a biochemical and physical comparison. Front Biosci. 1998 Jun 17;3:D570-603. Review. PMID: 9632377 2: Story RM, Weber IT, Steitz TA. The structure of the E. coli recA protein monomer and polymer. Nature. 1992 Jan 23;355(6358):318-25. Erratum in: Nature 1992 Feb 6;355(6360):567. PMID: 1731246 3: Story RM, Steitz TA. Structure of the recA protein-ADP complex. Nature. 1992 Jan 23;355(6358):374-6. PMID: 1731253 4: Story RM, Bishop DK, Kleckner N, Steitz TA. Structural relationship of bacterial RecA proteins to recombination proteins from bacteriophage T4 and yeast. Science. 1993 Mar 26;259(5103):1892-6. PMID: 8456313 5: Conway AB, Lynch TW, Zhang Y, Fortin GS, Fung CW, Symington LS, Rice PA. Crystal structure of a Rad51 filament. Nat Struct Mol Biol. 2004 Aug;11(8):791-6. Epub 2004 Jul 4. PMID: 15235592 6: Pellegrini L, Yu DS, Lo T, Anand S, Lee M, Blundell TL, Venkitaraman AR. Insights into DNA recombination from the structure of a RAD51-BRCA2 complex. Nature. 2002 Nov 21;420(6913):287-93. Epub 2002 Nov 10. PMID: 12442171

3. Holliday Model of Homologous Genetic Recombination

4. 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

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

6. 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: + +

7. 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.

8. 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

9. 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

10. RecA Filament Structure

11. Key Residues of E. coli RecA

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

13. Other Factors Involved in Homologous Recombination in E. coli (Mediators)

14. Conservation of RecA Family in Diverse Species

15. 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

16. Conservation of RecA Filament Structure RecA-dsDNA (ATPgS) T4 UvsX-dsDNA (ATP) Yeast Rad51-dsDNA (ADP-AlF4-)

17. Sequence Alignment of E. coli RecA, Yeast Rad51, & T4 UvsX

18. Modeling of Conserved Core Regions of T4 UvsX and Yeast Rad51 onto Known X-ray Structure of E. coli RecA

19. Identical Residues Mapped on E. coli RecA Structure UvsX vs. RecA Rad51 vs. RecA

20. Conservation of Other Recombination Functionalities (Mediators)

21. Crystal structure of a Rad51 filament. Conway AB, Lynch TW, Zhang Y, Fortin GS, Fung CW, Symington LS, Rice PA. Nat Struct Mol Biol. 2004 Aug;11(8):791-6.

22. N-terminally Truncated Yeast Rad51 (the form used for crystallization) Catalyzes Strand Exchange Crystallized form also contained I345T mutation-- a gain-of-function mutation, enhanced ssDNA-binding form, suppresses rad55/57 mediator mutations

23. Rad51 filament crystallized in presence of ssDNA oligo and ATPgS No ssDNA density. Loops disordered. SO42- occupies nucleotide binding site.

24. Rad51 filament crystallized in presence of ssDNA oligo and ATPgS Exact 3-fold but only approximate 6-fold screw symmetry. Alternating protomers are in different conformations! (Not seen in EcRecA structure)

25. Two Types of Protomer-Protomer Interactions at Rad51 ATPase Site

26. H352A Mutation Destabilizes Yeast Rad51-ssDNA Interactions Conserved His in Rad51 and RadA branches; Phe in bacterial RecAs Met in T-even UvsXs

27. Tyrosine Phosphorylation Site at Protomer-Protomer Interface

28. Relationship of BRCA Gene Products to Homologous Recombination & DSBR

29. Nobody Said It Would Be Simple…

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

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

32. 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

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

34. 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

35. The Rad51-BRC4 Interface Hydrophobic interactions via BRC4 a-helix Hydrophobic interactions via BRC4 b-hairpin Polar Interactions

36. Brca2 Inhibits Rad51 Filament Formation Crystallographic EcRecA Filament Superposition of BRC4 (from Rad51-BRC4 structure) on a subunit of EcRecA filament shows BRCA4 at interface between 2 EcRecA subunits. EcRecA sequence 26-IMRL-29 mediates polymerization by anti- parallel b-strand pairing Brca2 sequence 1524-FHTA-1527 interacts with Rad51 by anti- parallel b-strand pairing EcRecA interface Rad51-BRC4 interface

37. Yeast Rad51 Interface (Conway et al.) Resembles Brca2 Peptide Interaction with Monomeric Human Rad51 (Pellegrini et al.)

38. Mutant GFP-Fusion Rad51 Proteins F86E & A89E Designed to Disrupt Hydrophobic Contacts in Putative Rad51 Subunit Interface Result: WT but not mutant Rad51s can pull down endogenous WT Rad51 in co-IP expts. Ability to interact with Brca2 remains intact.

39. Impacts of Rad51 Polymerization Mutants F86E & A89E on Ability • to Form Nuclear Foci in Dividing 293T Cells or Following IR Exposure • Mutants inhibit foci formation • So does overexpression of BRC3/4 repeat fragment

40. Brca2: Designed to Load Rad51 Onto ssDNA?

41. Multiple BRC Repeats In Brca2 Could Serve as a Pre-Loading & Assembly Site for Rad51, All Ready for Transfer Onto ssDNA Bound to OB-folds in the DNA Binding Domain 3HB Motif in Tower Domain: Tether Complex to Duplex Portion of Tailed DSB??? Why Are Defects Mainly Associated With Tumors of Breast & Ovary???