In vivo assembly and disassembly of Rad51 and Rad52 complexes during double-strand break repair.
Assembly and disassembly of Rad51 and Rad52 complexes were monitored by immunofluorescence during homologous recombination initiated by an HO endonuclease-induced double-strand break (DSB) at the MAT locus. DSB-induced Rad51 and Rad52 foci colocalize with a TetR-GFP focus at tetO sequences adjacent to MAT. In strains in which HO cleaves three sites on chromosome III, we observe three distinct foci that colocalize with adjacent GFP chromosome marks. We compared the kinetics of focus formation with recombination intermediates and products when HO-cleaved MATalpha recombines with the donor, MATa. Rad51 assembly occurs 1 h after HO cleavage. Rad51 disassembly occurs at the same time that new DNA synthesis is initiated after single-stranded (ss) MAT DNA invades MATa. We present evidence for three distinct roles for Rad52 in recombination: a presynaptic role necessary for Rad51 assembly, a synaptic role with Rad51 filaments, and a postsynaptic role after Rad51 dissociates. Additional biochemical studies suggest the presence of an ssDNA complex containing both Rad51 and Rad52.
Miyazaki
Toshiko
T
Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan.
Bressan
Debra A
DA
Shinohara
Miki
M
Haber
James E
JE
Shinohara
Akira
A
D015825:Chromosomes, Fungal / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000235:genetics; D009154:Mutation; D051135:Rad51 Recombinase; D051721:Rad52 DNA Repair and Recombination Protein; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase; C530845:HO protein, S cerevisiae; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific