Heteroduplex formation by human Rad51 protein: effects of DNA end-structure, hRP-A and hRad52.
Purified human Rad51 protein (hRad51) catalyses ATP-dependent homologous pairing and strand transfer reactions, characteristic of a central role in homologous recombination and double-strand break repair. Using single-stranded circular and partially homologous linear duplex DNA, we found that the length of heteroduplex DNA formed by hRad51 was limited to approximately 1.3 kb, significantly less than that observed with Escherichia coli RecA and Saccharomyces cerevisiae Rad51 protein. Joint molecule formation required the presence of a 3' or 5'-overhang on the duplex DNA substrate and initiated preferentially at the 5'-end of the complementaryx strand. These results are consistent with a preference for strand transfer in the 3'-5' direction relative to the single-stranded DNA. The human single-strand DNA-binding protein, hRP-A, stimulated hRad51-mediated joint molecule formation by removing secondary structures from single-stranded DNA, a role similar to that played by E. coli single-strand DNA-binding protein in RecA-mediated strand exchange reactions. Indeed, E. coli single-strand DNA-binding protein could substitute for hRP-A in hRad51-mediated reactions. Joint molecule formation by hRad51 was stimulated or inhibited by hRad52, dependent upon the reaction conditions. The inhibitory effect could be overcome by the presence of hRP-A or excess heterologous DNA.
Baumann
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Clare Hall Laboratories, Imperial Cancer Research Fund, South Mimms, Hertfordshire, EN6 3LD, UK.
West
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D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes; D051135:Rad51 Recombinase; D051716:Replication Protein A; D013329:Structure-Activity Relationship
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009692:Nucleic Acid Heteroduplexes; C497167:RPA1 protein, human; D051716:Replication Protein A; C495153:RAD51 protein, human; D051135:Rad51 Recombinase