Preferential binding to branched DNA strands and strand-annealing activity of the human Rad51B, Rad51C, Rad51D and Xrcc2 protein complex

Yokoyama, Hiroshi; Sarai, Naoyuki; Kagawa, Wataru; Enomoto, Rima; Shibata, Takehiko; Kurumizaka, Hitoshi; Yokoyama, Shigeyuki

doi:10.1093/nar/gkh578

Cited by 82 publications

(75 citation statements)

References 61 publications

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“…The cells with the double knock-out of the RAD52 and XRCC2 proteins were significantly defective in the HRR pathway (61), indicating that the RAD52 protein has an overlapping function with the XRCC2 protein. The XRCC2 protein forms the BCDX2 complex, which is composed of the RAD51B, RAD51C, RAD51D, and XRCC2 proteins (62,63), and like the EVL and RAD52 proteins, the BCDX2 complex possesses robust ssDNA annealing activity (64). Therefore, redundant annealing activities may be required in the HRR pathway in higher eukaryotes.…”

Section: Discussionmentioning

confidence: 99%

Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL

Takaku

Machida

Hosoya

et al. 2009

Journal of Biological Chemistry

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The RAD51 protein is a central player in homologous recombinational repair. The RAD51B protein is one of five RAD51 paralogs that function in the homologous recombinational repair pathway in higher eukaryotes. In the present study, we found that the human EVL (Ena/Vasp-like) protein, which is suggested to be involved in actin-remodeling processes, unexpectedly binds to the RAD51 and RAD51B proteins and stimulates the RAD51-mediated homologous pairing and strand exchange. The EVL knockdown cells impaired RAD51 assembly onto damaged DNA after ionizing radiation or mitomycin C treatment. The EVL protein alone promotes single-stranded DNA annealing, and the recombination activities of the EVL protein are further enhanced by the RAD51B protein. The expression of the EVL protein is not ubiquitous, but it is significantly expressed in breast cancer-derived MCF7 cells. These results suggest that the EVL protein is a novel recombination factor that may be required for repairing specific DNA lesions, and that may cause tumor malignancy by its inappropriate expression.Chromosomal DNA double strand breaks (DSBs) 2 are potential inducers of chromosomal aberrations and tumorigenesis, and they are accurately repaired by the homologous recombinational repair (HRR) pathway, without base substitutions, deletions, and insertions (1-3). In the HRR pathway (4, 5), single-stranded DNA (ssDNA) tails are produced at the DSB sites. The RAD51 protein, a eukaryotic homologue of the bacterial RecA protein, binds to the ssDNA tail and forms a helical nucleoprotein filament. The RAD51-ssDNA filament then binds to the intact double-stranded DNA (dsDNA) to form a threecomponent complex, containing ssDNA, dsDNA, and the RAD51 protein. In this three-component complex, the RAD51 protein promotes recombination reactions, such as homologous pairing and strand exchange (6 -9).The RAD51 protein requires auxiliary proteins to promote the homologous pairing and strand exchange reactions efficiently in cells (10 -12). In humans, the RAD52, RAD54, and RAD54B proteins directly interact with the RAD51 protein (13-17) and stimulate the RAD51-mediated homologous pairing and/or strand exchange reactions in vitro (18 -21). The human RAD51AP1 protein, which directly binds to the RAD51 protein (22), was also found to stimulate RAD51-mediated homologous pairing in vitro (23, 24). The BRCA2 protein contains ssDNA-binding, dsDNA-binding, and RAD51-binding motifs (25)(26)(27)(28)(29)(30)(31)(32)(33), and the Ustilago maydis BRCA2 ortholog, Brh2, reportedly stimulated RAD51-mediated strand exchange (34,35). Most of these RAD51-interacting factors are known to be required for efficient RAD51 assembly onto DSB sites in cells treated with ionizing radiation (10 -12).The RAD51B (RAD51L1, Rec2) protein is a member of the RAD51 paralogs, which share about 20 -30% amino acid sequence similarity with the RAD51 protein (36 -38). RAD51B-deficient cells are hypersensitive to DSB-inducing agents, such as cisplatin, mitomycin C (MMC), and ␥-rays, indicating that the RAD51B protei...

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Section: Discussionmentioning

confidence: 99%

Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL

Takaku

Machida

Hosoya

et al. 2009

Journal of Biological Chemistry

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“…The Rad51C-Xrcc3 complex has been associated with Holliday junction resolvase activities in mammalian cells (21). In addition, the BCDX2 complex has a preference for binding to branched DNA structures (19,(21)(22)(23)) and a role for Xrcc2 or Xrcc3 in replication fork progression after DNA damage has also been proposed (24). Thus the role of Rad51 paralogs in HRR is complicated and likely depends on the different functional complexes of the paralogs and their interacting partners.…”

Section: Homologous Recombination Repair (Hrr)mentioning

confidence: 99%

“…However, Rad51B and the BCDX2 complex have been shown to bind to branched structures including Y-shaped molecules and Holliday junctions as demonstrated by electrophoretic mobility shift assays (EMSA) (20,23,34). Visualization of binding to Holliday junctions and forked DNA structures by EM however, has up until now, been lacking.…”

Section: Proteinmentioning

confidence: 99%

Ring-shaped Rad51 Paralog Protein Complexes Bind Holliday Junctions and Replication Forks as Visualized by Electron Microscopy

Compton

Özgür

Griffith

2010

Journal of Biological Chemistry

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In mammals, there are five Rad51 paralogs that form two distinct complexes in vivo. One complex is composed of Rad51B-Rad51C-Rad51D-Xrcc2 (BCDX2) and the other Rad51C-Xrcc3 (CX3). We co-expressed and purified human BCDX2 and CX3 protein complexes from insect cells and investigated their binding preferences and structure using transmission electron microscopy (TEM). We visualized the binding of BCDX2 and CX3 to DNA templates containing replication forks and Holliday junctions, intermediates observed during DNA replication and recombination, respectively. We show that both complexes bind with exceptionally high specificity to the DNA junctions with little binding observed elsewhere on the DNAs. Further analysis of the structure of free or DNA-bound BCDX2 and CX3 complexes revealed a multimeric ring structure whose subunits are arranged into a flat disc around a central channel. This work provides the first EM visualization of BCDX2 and CX3 binding to Holliday junctions and forked DNAs and suggests the complexes form ring-shaped structures.

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“…Rad51B and the BCDX2 complex have been shown to preferentially bind synthetic Holliday junctions (HJs) over other types of DNA substrates (Yokoyama et al 2004). Furthermore, extracts made from XRCC3 À/À or RAD51C À/À hamster cells lacked normal levels of HJ resolvase activity, suggesting that the Rad51C-Xrcc3 complex may contribute to the resolution of recombination intermediates (Liu et al 2004).…”

mentioning

confidence: 99%

Role of the Saccharomyces cerevisiae Rad51 Paralogs in Sister Chromatid Recombination

2008

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Rad51 requires a number of other proteins, including the Rad51 paralogs, for efficient recombination in vivo. Current evidence suggests that the yeast Rad51 paralogs, Rad55 and Rad57, are important in formation or stabilization of the Rad51 nucleoprotein filament. To gain further insights into the function of the Rad51 paralogs, reporters were designed to measure spontaneous or double-strand break (DSB)-induced sister or nonsister recombination. Spontaneous sister chromatid recombination (SCR) was reduced 6000-fold in the rad57 mutant, significantly more than in the rad51 mutant. Although the DSBinduced recombination defect of rad57 was suppressed by overexpression of Rad51, elevated temperature, or expression of both mating-type alleles, the rad57 defect in spontaneous SCR was not strongly suppressed by these same factors. In addition, the UV sensitivity of the rad57 mutant was not strongly suppressed by MAT heterozygosity, even though Rad51 foci were restored under these conditions. This lack of suppression suggests that Rad55 and Rad57 have different roles in the recombinational repair of stalled replication forks compared with DSB repair. Furthermore, these data suggest that most spontaneous SCR initiates from single-stranded gaps formed at stalled replication forks rather than DSBs.

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Preferential binding to branched DNA strands and strand-annealing activity of the human Rad51B, Rad51C, Rad51D and Xrcc2 protein complex

Cited by 82 publications

References 61 publications

Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL

Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL

Ring-shaped Rad51 Paralog Protein Complexes Bind Holliday Junctions and Replication Forks as Visualized by Electron Microscopy

Role of the Saccharomyces cerevisiae Rad51 Paralogs in Sister Chromatid Recombination

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