Olga M. Mazina

RAD54 PROTEIN PROMOTES BRANCH MIGRATION OF HOLLIDAY JUNCTIONS

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.

Rad54, the motor of homologous recombination

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.

Inhibition of homologous recombination in human cells by targeting RAD51 recombinase.

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.

The resistance of DMC1 D-loops to dissociation may account for the DMC1 requirement in meiosis

The ubiquitously expressed Rad51 recombinase and the meiosis-specific Dmc1 recombinase promote the formation of strand-invasion products (D-loops) between homologous molecules. Strand-invasion products are processed by either the double-strand break repair (DSBR) or synthesis-dependent strand annealing (SDSA) pathway. D-loops destined to be processed by SDSA need to dissociate, producing non-crossovers, and those destined for DSBR should resist dissociation to generate crossovers. The mechanism that channels recombination intermediates into different homologous-recombination pathways is unknown. Here we show that D-loops in a human DMC1-driven reaction are substantially more resistant to dissociation by branch-migration proteins such as RAD54 than those formed by RAD51. We propose that the intrinsic resistance to dissociation of DMC1 strand-invasion intermediates may account for why DMC1 is essential to ensure the proper segregation of chromosomes in meiosis.

Bloom Syndrome Helicase Stimulates RAD51 DNA Strand Exchange Activity through a Novel Mechanism

Loss or inactivation of BLM, a helicase of the RecQ family, causes Bloom syndrome, a genetic disorder with a strong predisposition to cancer. Although the precise function of BLM remains unknown, genetic data has implicated BLM in the process of genetic recombination and DNA repair. Previously, we demonstrated that BLM can disrupt the RAD51-single-stranded DNA filament that promotes the initial steps of homologous recombination. However, this disruption occurs only if RAD51 is present in an inactive ADP-bound form. Here, we investigate interactions of BLM with the active ATP-bound form of the RAD51-single-stranded DNA filament. Surprisingly, we found that BLM stimulates DNA strand exchange activity of RAD51. In contrast to the helicase activity of BLM, this stimulation does not require ATP hydrolysis. These data suggest a novel BLM function that is stimulation of the RAD51 DNA pairing. Our results demonstrate the important role of the RAD51 nucleoprotein filament conformation in stimulation of DNA pairing by BLM.

Human Rad54 protein stimulates human Mus81–Eme1 endonuclease

Rad54, a key protein of homologous recombination, physically interacts with a DNA structure-specific endonuclease, Mus81–Eme1. Genetic data indicate that Mus81–Eme1 and Rad54 might function together in the repair of damaged DNA. In vitro, Rad54 promotes branch migration of Holliday junctions, whereas the Mus81–Eme1 complex resolves DNA junctions by endonucleolytic cleavage. Here, we show that human Rad54 stimulates Mus81–Eme1 endonuclease activity on various Holliday junction-like intermediates. This stimulation is the product of specific interactions between the human Rad54 (hRad54) and Mus81 proteins, considering that Saccharomyces cerevisiae Rad54 protein does not stimulate human Mus81–Eme1 endonuclease activity. Stimulation of Mus81–Eme1 cleavage activity depends on formation of specific Rad54 complexes on DNA substrates occurring in the presence of ATP and, to a smaller extent, of other nucleotide cofactors. Thus, our results demonstrate a functional link between the branch migration activity of hRad54 and the structure-specific endonuclease activity of hMus81–Eme1, suggesting that the Rad54 and Mus81–Eme1 proteins may cooperate in the processing of Holliday junction-like intermediates during homologous recombination or DNA repair.

A role for SSRP1 in recombination‐mediated DNA damage response

A possible role for structure‐specific recognition protein 1 (SSRP1) in replication‐associated repair processes has previously been suggested based on its interaction with several DNA repair factors and the replication defects observed in SSRP1 mutants. In this study, we investigated the potential role of SSRP1 in association with DNA repair mediated by homologous recombination (HR), one of the pathways involved in repairing replication‐associated DNA damage, in mammalian cells. Surprisingly, over‐expression of SSRP1 reduced the number of hprt+ recombinants generated via HR both spontaneously and upon hydroxyurea (HU) treatment, whereas knockdown of SSRP1 resulted in an increase of HR events in response to DNA double‐strand break formation. In correlation, we found that the depletion of SSRP1 in HU‐treated human cells elevated the number of Rad51 and H2AX foci, while over‐expression of the wild‐type SSRP1 markedly reduced HU‐induced Rad51 foci formation. We also found that SSRP1 physically interacts with a key HR repair protein, Rad54 both in vitro and in vivo. Further, branch migration studies demonstrated that SSRP1 inhibits Rad54‐promoted branch migration of Holliday junctions in vitro. Taken together, our data suggest a functional role for SSRP1 in spontaneous and replication‐associated DNA damage response by suppressing avoidable HR repair events. J. Cell. Biochem. 108: 508–518, 2009.

HOP2-MND1 modulates RAD51 binding to nucleotides and DNA

The HOP2-MND1 heterodimer is required for progression of homologous recombination in eukaryotes. In vitro, HOP2-MND1 stimulates the DNA strand exchange activities of RAD51 and DMC1. We demonstrate that HOP2-MND1 induces changes in the conformation of RAD51 that profoundly alter the basic properties of RAD51. HOP2-MND1 enhances the interaction of RAD51 with nucleotide cofactors and modifies its DNA binding specificity in a manner that stimulates DNA strand exchange. It enables RAD51 DNA strand exchange in the absence of divalent metal ions required for ATP binding and offsets the effect of the K133A mutation that disrupts ATP binding. During nucleoprotein formation HOP2-MND1 helps to load RAD51 on ssDNA restricting its dsDNA-binding and during the homology search it promotes dsDNA binding removing the inhibitory effect of ssDNA. The magnitude of the changes induced in RAD51 defines HOP2-MND1 as a “molecular trigger” of RAD51 DNA strand exchange.

Targeting BRCA1- and BRCA2-deficient cells with RAD52 small molecule inhibitors

RAD52 is a member of the homologous recombination (HR) pathway that is important for maintenance of genome integrity. While single RAD52 mutations show no significant phenotype in mammals, their combination with mutations in genes that cause hereditary breast cancer and ovarian cancer like BRCA1, BRCA2, PALB2 and RAD51C are lethal. Consequently, RAD52 may represent an important target for cancer therapy. In vitro, RAD52 has ssDNA annealing and DNA strand exchange activities. Here, to identify small molecule inhibitors of RAD52 we screened a 372,903-compound library using a fluorescence-quenching assay for ssDNA annealing activity of RAD52. The obtained 70 putative inhibitors were further characterized using biochemical and cell-based assays. As a result, we identified compounds that specifically inhibit the biochemical activities of RAD52, suppress growth of BRCA1- and BRCA2-deficient cells and inhibit RAD52-dependent single-strand annealing (SSA) in human cells. We will use these compounds for development of novel cancer therapy and as a probe to study mechanisms of DNA repair.

Polarity and Bypass of DNA Heterology during Branch Migration of Holliday Junctions by Human RAD54, BLM, and RECQ1 Proteins

Background: Several proteins catalyze branch migration (BM) of the Holliday junction. Results: RAD54 is a robust BM protein capable of bypassing extensive regions of DNA heterology. RAD54, BLM, and RECQ1 drive BM in the 3′→5′ direction. Conclusion: The displacement strand of joint molecules (JMs) defines the polarity of BM. Significance: BM is mechanistically distinct from helicase activity of DNA translocating proteins. Several proteins have been shown to catalyze branch migration (BM) of the Holliday junction, a key intermediate in DNA repair and recombination. Here, using joint molecules made by human RAD51 or Escherichia coli RecA, we find that the polarity of the displaced ssDNA strand of the joint molecules defines the polarity of BM of RAD54, BLM, RECQ1, and RuvAB. Our results demonstrate that RAD54, BLM, and RECQ1 promote BM preferentially in the 3′→5′ direction, whereas RuvAB drives it in the 5′→3′ direction relative to the displaced ssDNA strand. Our data indicate that the helicase activity of BM proteins does not play a role in the heterology bypass. Thus, RAD54 that lacks helicase activity is more efficient in DNA heterology bypass than BLM or REQ1 helicases. Furthermore, we demonstrate that the BLM helicase and BM activities require different protein stoichiometries, indicating that different complexes, monomers and multimers, respectively, are responsible for these two activities. These results define BM as a mechanistically distinct activity of DNA translocating proteins, which may serve an important function in DNA repair and recombination.