Mechanisms of Homologous Recombination & Protein

Rad51

Jia LiuAdam Jara

Jan 20th, 2011

MCB 720

Outline• I. Introduction• II. Mechanisms of Homologous

Recombination (HR)• III. HR in Saccharomyces cerevisiae

(SC);• IV. Rad 51 protein;• V. Conclusion.

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.

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.

Heyer W, Ehmsen KT, Jie L. (2010) Annu. Rev. Genetics 44: 113-39

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

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

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

Double Holliday Junction (dHJ)

Helleday T, Lo J, van Gent D, Engelward B. (2007) DNA Repair 6: 923-35.

•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.

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.

Major Proteins Involved in HR

Hinz J. (2010) Environmental and Molecular Mutagenesis 51:582-603.

•HR requires 3’ overhang that constitutes the presynaptic filament

•DNA Pol is resposible for forming the completary strands

•Holliday heteroduplexes must be resolved.

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.

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.

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).

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.

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” .

C. Interacting partners

Homologous recombination factors in Saccharomyces cerevisiae

S. Cerevisiae

Biochemical activity/function Interacting partners

Rad51 ATP-dependent DNA-binding activity; ATP-dependent homologous pairing and DNA strand exchange activities

Yeast: Rad52, Rad54, Rad55,Rdh54/Tid1, Sgs1, Rsi1/Apc2, Zip3,Dmc1*, Mlh1, Sap1, Ubc9, YMR233W,YPL238C, YPR011C; human: Rad51C,Rad52, Rad54, Rad54B*, Werner’ssyndrome protein, Xrcc3, c-Abl, p53,Brca1*, Brca2, Ube2I/Ubc9, Ubl1,Pir51, Rpb1.

Dudas A, Chovanec M. (2004) Mutation Research 566: 131-167.

Protein–protein interaction network

(A) mitosis (B) meiosis

Dudas A, Chovanec M. (2004) Mutation Research 566: 131-167.

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.

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.