Mechanisms of Homologous Recombination & Protein Rad51

1. MCB 720 Mechanisms of Homologous Recombination & Protein Rad51 Jia Liu Adam Jara Jan 20th, 2011

2. Outline • I. Introduction • II. Mechanisms of Homologous Recombination (HR) • III. HR in Saccharomyces cerevisiae (SC); • IV. Rad 51 protein; • V. Conclusion.

3. I. Introduction • HR allows for proper chromosome segregation during meiosis, promoting genomic integrity between generations. • Represents the highest fidelity repair mechanism for DNA breaks. • Diploid human cell maintains ~6x109 bp of DNA. • Highly regulated process, as too much HR can lead to large chromosomal rearrangements.

4. Double Strand Break Repair • Unintended DSB represents on of the most toxic DNA lesions and can result in chromosomal rearrangement. • These can occur from environmental stresses: ie. ionizing radiation, pharmaceutics. • Mammalian cells have three main mechanisms of DSB repair: non-homologous end joining, single strand annealing, and HR.

5. Pathways of DSB Repair • The primary pathway in eukaryotes for repairing DSB is NHEJ • NHEJ can lead to gross genetic rearrangment and loss of genomic material • SSA depends on tandem repeats and can also lead to loss of genomic material • Only HR offers the capacity to resynthesize damaged or missing sequence. A template is necessary Heyer W, Ehmsen KT, Jie L. (2010) Annu. Rev. Genetics 44: 113-39

6. Regulation of Homologous Recombination • During S phase sister chromatids are present and are the best substrate for HR • CDKs present during S phase phosphorylate CtIP. • CtIP mediates 5’ end resection allowing for 3’ overhangs • Unphosphorylated BRCA2 no longer inhibits Rad51. • Rad51 forms nucleofilaments with the 3’ overhangs and promotes HR. Heyer W, Ehmsen KT, Jie L. (2010) Annu. Rev. Genetics 44: 113-39

7. Synthesis Dependent Strand Annealing (SDSA) • Principal mechanism for DSB repair by HR. Reduces the potential for risk of crossover. • The MRN (MRE11, NBS1, RAD50) complex recognizes the DSB and processes the 3’ overhang which is initially bound by RPA. • RAD52 and BRCA2 mediate RPA dissociation and RAD51 nucleofilament formation. • RAD51 performs strand invasion and homology search forming a displacement (D-) loop. • DNA is synthesized by DNA Pol η (mammals) • New strand dissociates and binds to original 3’ end. Gap is filled by DNA ligase. Lodish et al., Molecular Cell Biology, 5th ed. Fig 23-31

8. Double Holliday Junction (dHJ) • Most likely mechanism by which crossover occurs. • Involves the strand invasion by both 3’ RAD51 nucleofilaments. • Resolution of the heteroduplexes (Holliday Junctions) by many different endonucleases (MUS81-EME1 , GEN1, TOP1). • Directionality of resolution determines gene conversion or crossover. Helleday T, Lo J, van Gent D, Engelward B. (2007) DNA Repair 6: 923-35.

9. Loss of HR Regulation • BRCA2 facilitates displacement of RPA and loading of Rad51 onto ssDNA. • Loss of function leads to chromosome abnormalities. • BLM functions as a 3’->5’ helicase in presynaptic events • Mutants lead to Bloom’s Syndrome, a condition characterized by a predisposition to cancer and loss of heterozygosity.

10. Major Proteins Involved in HR • HR requires 3’ overhang that constitutes the presynaptic filament • DNA Pol is resposible for forming the completary strands • Holliday heteroduplexes must be resolved. Hinz J. (2010) Environmental and Molecular Mutagenesis 51:582-603.

11. III. HR in Saccharomyces cerevisiae (SC) • Saccharomyces cerevisiae; • RAD 52 epistasis group: RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, RDH54/TID1, MRE11 and XRS2 genes; • RAD 51 family; • RAD51: wild type gene or locus; rad51: the mutant gene or locus; Rad51: protein. http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae, accessed on Jan 17th, 2011. Alan Wheals. Scanning Electron Micrograph of Saccharomyces cerevisiae.

12. IV. Rad 51 in HR of S. cerevisiae A. Mutant phenotype • S. cerevisiae rad51 mutants; • Sensitive to IR; reduce mitotic and meiotic recombination; • Chemical agents; anticanter drugs; • Affect recombination events; • Meiosis.

13. B. Gene and protein • ScRad51; • 400 A.A., 43kDa; • 3 regions: N terminal, C terminal, central core; • A putative leucine zipper motif; • Rad51-dependent pathways: DSB-induced gene conversion; • Rad51-independent pathways: spontaneous recombination, DSB-induced DNA replication (BIR).

14. ScRad51: Homologue of Rec A • Structures similarities; • Main RecA-like recombinase; • ATP-dependent DNA-binding activity; • Difference: Requirements for initiating joint molecules and the polarity of branch migration. • Rec A: ssDNA; • ScRad 51:dsDNA with ssDNA tails.

15. C. Interacting partners • Interactions with multiple proteins; • ScRad 51: ScRad 51, Rad52, Rad 54, Rad55, Rdh54/Tid1(Rad54), Sgs1. • Complex involving a large number of proteins “recombinosome” .

16. C. Interacting partners Homologous recombination factors in Saccharomyces cerevisiae Dudas A, Chovanec M. (2004) Mutation Research 566: 131-167.

17. Protein–protein interaction network (A) mitosis (B) meiosis Dudas A, Chovanec M. (2004) Mutation Research 566: 131-167.

18. V. Conclusion • HR is the pathway by which genome integrity is maintained • High fidelity pathway for DSB repair • Drive genetic recombination • The main HR pathway involved in DSB repair is SDSA • The dHJ pathway likely allows for mitotic crossover events • The protein involved in eukaryotes HR is Rad51 (RecA homolog), which forms complex with multiple proteins, promoting homologous pairing and DNA strand exchange activities in HR.

19. References • Dudas A, Chovanec M. (2004) DNA double-strand break repair by homologous recombination. Mutation Research 566: 131-167. • Helleday T, Lo J, van Gent DC, Engelward BP. (2007) DNA double-strand break repair: From mechanistic understanding to cancer treatment. DNA Repair 6: 923-935 • Heyer W, Ehmsen KT, Liu J. (2010) Regulation of Homologous Recombination in Eukaryotes. Annu. Rev. Genet. 44: 113-39. • Hinz JM. (2010) Role of Homologous Recombination in DNA Interstrand Crosslink Repair. Environmental and Molecular Mutagenesis 51: 582-603.