Rad5l protein of Saccharomyces cerevisiae is a structural homolog of the Escherichia coli recombination enzyme RecA. In yeast, the Rad5l protein is required for mitotic and meiotic recombination and for repair of doublestrand breaks in DNA. We have used antibodies raised against the homologous human protein, HsRad51, expressed

A sensitive and rapid in situ method was developed to visualize sites of single-stranded (ss) DNA in cultured cells and in experimental test animals. Anti-bromodeoxyuridine antibody recognizes the halogenated base analog incorporated into chromosomal DNA only when substituted DNA is in the single strand form. After treatment of cells with DNA-damaging agents or ␥ irradiation, ssDNA molecules form nuclear foci in a dose-dependent manner within 60 min. The mammalian recombination protein Rad51 and the replication protein A then accumulate at sites of ssDNA and form foci, suggesting that these are sites of recombinational DNA repair.Eukaryotic cells are endowed with multiple pathways to repair damaged DNA. One of the major pathways is nucleotide excision repair, which can remove a broad range of DNA lesions. Nucleotide excision repair excises oligonucleotides of 25-32 bp including the damaged DNA, filling in the single-stranded (ss) DNA gap (1). This process is very efficient and usually repairs most DNA lesions before the damaged region is replicated. However, if the replication fork meets unrepaired DNA damage, breaks may occur in one or both strands of the nascent DNA (2). Single-and double-strand breaks (DSBs) may also arise as a direct effect of many DNA-damaging agents such as ionizing radiation. Singlestrand breaks are efficiently repaired and do not represent a threat for cell survival. In contrast, DSBs are potentially lethal. DSBs may be repaired either by direct end-joining of the broken ends in a process that is mediated by the Mre11-Rad50 complex or by homologous recombination (3). In yeast, DSBs are mainly repaired by homologous recombination; genes of the Rad52 epistasis group are largely responsible for this pathway. Although mammalian cells are presumed to repair DSBs predominantly by nonhomologous end-joining (4, 5), accumulating experimental evidence suggests that homologous recombination also plays an essential role in mammalian DSB repair (6-8).DSB repair by homologous recombination starts with 5Ј to 3Ј-exonucleolytic digestion of one DNA strand, which leads to the formation of 3Ј-overhanging ssDNA tails. In Escherichia coli, this ssDNA associates with the RecA recombination enzyme, forming the ssDNA-RecA filament. This RecA-nucleoprotein filament promotes homology search by the single DNA strand and initiates the exchange between ssDNA and homologous double-stranded (ds) DNA (3, 9). Rad51 is a structural and functional eukaryotic homolog of E. coli RecA (10, 11). Similar to RecA, both yeast and mammalian Rad51 proteins form nucleoprotein filaments on ssDNA, mediating homologous pairing and strand-exchange reactions between ssDNA and homologous dsDNA (12-15). Homologous pairing and DNA-strand exchange mediated by Rad51 are facilitated in vitro by the ssDNA-binding protein, RPA (12,(16)(17)(18).In normal, cultured human cells, the HsRad51 protein is detected in multiple discrete foci in the nucleoplasm of a low number of cells by immunofluorescent antibodies. After DNA damage the percent...

Homologous pairing and strand exchange, which are catalyzed by Escherichia coli RecA protein, are central to homologous recombination. Homologs of this protein are found in eukaryotes; however, little has been reported on the recombinase activities of the mammalian homologs, including the human protein, denoted HsRad51. For the studies described here, we purified HsRad51 from E. coli. Although the activities of HsRad51 and RecA were qualitatively similar in the presence of ATP, there were also striking differences. The stoichiometry of binding to DNA and the rate of renaturation of complementary strands were similar for the two proteins, but rates of ATP hydrolysis, homologous pairing, and subsequent strand exchange promoted by HsRad51 were less than 1 ⁄10 those of RecA. In addition, HsRad51 bound ␥-thio-ATP and formed stable presynaptic complexes that promoted renaturation as rapidly as RecA, but the recombinant human protein catalyzed neither strand exchange nor homologous pairing of a single strand with duplex DNA in the presence of the ATP analog. By contrast, RecA promoted both of the latter reactions in control experiments. These observations suggest that among RecA-like proteins, HsRad51 may be a variant in which homologous pairing and strand exchange are more closely linked to the hydrolysis of ATP.

Replication protein A (RPA), a heterotrimeric single-stranded DNA binding protein, is required for recombination, and stimulates homologous pairing and DNA strand exchange promoted in vitro by human recombination protein HsRad51. Co-immunoprecipitation revealed that purified RPA interacts physically with HsRad51, as well as with HsDmc1, the homolog that is expressed specifically in meiosis. The interaction with HsRad51 was mediated by the 70 kDa subunit of RPA, and according to experiments with deletion mutants, this interaction required amino acid residues 169-326. In exponentially growing mammalian cells, 22% of nuclei showed foci of RPA protein and 1-2% showed foci of Rad51. After gamma-irradiation, the percentage of cells with RPA foci increased to approximately 50%, and those with Rad51 foci to 30%. All of the cells with foci of Rad51 had foci of RPA, and in those cells the two proteins co-localized in a high fraction of foci. The interactions of human RPA with Rad51, replication proteins and DNA are suited to the linking of recombination to replication.