Homologous recombination has a crucial function in the repair of DNA double-strand breaks and in faithful chromosome segregation. The mechanism of homologous recombination involves the search for homology and invasion of the ends of a broken DNA molecule into homologous duplex DNA to form a cross-stranded structure, a Holliday junction (HJ). A HJ is able to undergo branch migration along DNA, generating increasing or decreasing lengths of heteroduplex. In both prokaryotes and eukaryotes, the physical evidence for HJs, the key intermediate in homologous recombination, was provided by electron microscopy. In bacteria there are specialized enzymes that promote branch migration of HJs. However, in eukaryotes the identity of homologous recombination branch-migration protein(s) has remained elusive. Here we show that Rad54, a Swi2/Snf2 protein, binds HJ-like structures with high specificity and promotes their bidirectional branch migration in an ATPase-dependent manner. The activity seemed to be conserved in human and yeast Rad54 orthologues. In vitro, Rad54 has been shown to stimulate DNA pairing of Rad51, a key homologous recombination protein. However, genetic data indicate that Rad54 protein might also act at later stages of homologous recombination, after Rad51 (ref. 13). Novel DNA branch-migration activity is fully consistent with this late homologous recombination function of Rad54 protein.

Homologous recombination (HR) performs crucial functions including DNA repair, segregation of homologous chromosomes, propagation of genetic diversity, and maintenance of telomeres. HR is responsible for the repair of DNA double-strand breaks and DNA interstrand cross-links. The process of HR is initiated at the site of DNA breaks and gaps and involves a search for homologous sequences promoted by Rad51 and auxiliary proteins followed by the subsequent invasion of broken DNA ends into the homologous duplex DNA that then serves as a template for repair. The invasion produces a cross-stranded structure, known as the Holliday junction. Here, we describe the properties of Rad54, an important and versatile HR protein that is evolutionarily conserved in eukaryotes. Rad54 is a motor protein that translocates along dsDNA and performs several important functions in HR. The current review focuses on the recently identified Rad54 activities which contribute to the late phase of HR, especially the branch migration of Holliday junctions.

SUMMARY RNA can serve as a template for DNA double-strand break repair in yeast cells, and Rad52, a member of the homologous recombination pathway, emerged as an important player in this process. However, the exact mechanism of how Rad52 contributes to RNA-dependent DSB repair remained unknown. Here, we report an unanticipated activity of yeast and human Rad52: inverse strand exchange, in which Rad52 forms a complex with dsDNA and promotes strand exchange with homologous ssRNA or ssDNA. We show that in eukaryotes, inverse strand exchange between homologous dsDNA and RNA is a distinctive activity of Rad52; neither Rad51 recombinase nor the yeast Rad52 paralog Rad59 has this activity. In accord with our in vitro results, our experiments in budding yeast provide evidence that Rad52 inverse strand exchange plays an important role in RNA-templated DSB repair in vivo.

The homologous recombination (HR) pathway plays a crucial role in the repair of DNA double-strand breaks (DSBs) and interstrand cross-links (ICLs). RAD51, a key protein of HR, possesses a unique activity: DNA strand exchange between homologous DNA sequences. Recently, using a high-throughput screening (HTS), we identified compound 1 (B02), which specifically inhibits the DNA strand exchange activity of human RAD51. Here, we analyzed the mechanism of inhibition and found that 1 disrupts RAD51 binding to DNA. We then examined the effect of 1 on HR and DNA repair in the cell. The results show that 1 inhibits HR and increases cell sensitivity to DNA damage. We propose to use 1 for analysis of cellular functions of RAD51. Because DSB- and ICL-inducing agents are commonly used in anticancer therapy, specific inhibitors of RAD51 may also help to increase killing of cancer cells.

Mutations in the Drosophila mus308 gene confer specific hypersensitivity to DNA-cross-linking agents as a consequence of defects in DNA repair. The mus308 gene is shown here to encode a 229-kDa protein in which the amino-terminal domain contains the seven conserved motifs characteristic of DNA and RNA helicases and the carboxy-terminal domain shares over 55% sequence similarity with the polymerase domains of prokaryotic DNA polymerase I-like enzymes. This is the first reported member of this family of DNA polymerases in a eukaryotic organism, as well as the first example of a single polypeptide with homology to both DNA polymerase and helicase motifs. Identification of a closely related gene in the genome of Caenorhabditis elegans suggests that this novel polypeptide may play an evolutionarily conserved role in the repair of DNA damage in eukaryotic organisms.