<b>Purification and characterization of XRad51.1 protein, <i>Xenopus RAD51</i> homologue: recombinant XRad51.1 promotes strand exchange reaction</b>

Maeshima, Kazuhiro; Morimatsu, Katsumi; Horii, Toshihiro

doi:10.1046/j.1365-2443.1996.d01-224.x

Cited by 36 publications

(36 citation statements)

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“…In previous studies, observations on the interaction of fluorescent probes revealed that homologous pairing, the initial formation of homologously aligned joint molecules is much slower when catalyzed by human Rad51 protein than by RecA protein. Strand exchange itself, as detected specifically by the separation of contiguous fluorescent probes, is also very slow in the case of HsRad51 (23,38), as are the overall reactions promoted by ScRad51 (20), HsRad51 (22), Xrad51 (21), and HsDmc1 (this paper, see above).…”

Section: Discussionmentioning

confidence: 83%

“…Strand exchange activities have been reported for Rad51 from S. cerevisiae, Xenopus laevis, and Homo sapiens (20)(21)(22)(23), but none has been reported for Dmc1. Other studies show that both Rad51 and Dmc1 associate with synaptonemal complexes in the mouse; but according to the location and time of appearance of the two proteins during meiosis, Rad51 and Dmc1 appear to play different roles (24,25).…”

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confidence: 99%

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Recombination activities of HsDmc1 protein, the meiotic human homolog of RecA protein

Golub

Gupta

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

132

116

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Meiosis-specific homologs of RecA protein have been identified in Saccharomyces cerevisiae and higher eukaryotes including mammals, but their enzymatic activities have not been described. We have purified the human protein HsDmc1 produced in Escherichia coli from a cloned copy of the cDNA. The recombinant enzyme had DNA-dependent ATPase activity with an estimated k cat of 1.5 min ؊1 . DNase protection experiments with oligonucleotides as substrates indicated that HsDmc1 protein binds preferentially to single-stranded DNA with a stoichiometry of approximately one molecule of protein per three nucleotide residues. HsDmc1 protein catalyzed the formation of D-loops in superhelical DNA, as well as strand exchange between single-stranded and double-stranded oligonucleotides. The requirements for strand exchange catalyzed by HsDmc1 were similar to those of RecA protein, but exchange caused by HsDmc1 was not supported by ATP␥S.

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Section: Discussionmentioning

confidence: 83%

mentioning

confidence: 99%

Recombination activities of HsDmc1 protein, the meiotic human homolog of RecA protein

Golub

Gupta

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

132

116

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“…In bacteria, RecA is the recombinase that catalyzes homologous pairing (Shibata et al, 1979;McEntee et al, 1979). In eukaryotes, two recombinases, RAD51 (denoted as Rad51 in Saccharomyces cerevisiae) and DMC1 (denoted as Dmc1 in S. cerevisiae), which are homologues of RecA (Aboussekhra et al, 1992;Basile et al, 1992;Bishop et al, 1992;Shinohara et al, 1992Shinohara et al, , 1993Habu et al, 1996), catalyze homologous pairing (Sung, 1994;Baumann et al, 1996;Maeshima et al, 1996;Gupta et al, 1997;Li et al, 1997;Hong et al, 2001). The mammalian and yeast genomes contain one copy each of the RAD51 and DMC1 genes (Bishop et al, 1992;Shinohara et al, 1992Shinohara et al, , 1993Habu et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

Green fluorescent protein fused to the C terminus of RAD51 specifically interferes with secondary DNA binding by the RAD51-ssDNA complex

et al. 2014

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Green fluorescent protein (GFP), fused to the N or C terminus of a protein of interest, is widely used to monitor the localization and mobility of proteins in cells. RAD51 is an essential protein that functions in mitotic DNA repair and meiotic chromosome segregation by promoting the homologous recombination reaction. A previous genetic study with Arabidopsis thaliana revealed that GFP fused to the C terminus of RAD51 (RAD51-GFP) inhibits mitotic DNA repair, but meiotic homologous recombination remained unaffected. To determine how the C-terminal GFP specifically inhibits mitotic DNA repair by RAD51, we purified rice RAD51A1-GFP and RAD51A2-GFP, and performed biochemical analyses. Interestingly, purified RAD51A1-GFP and RAD51A2-GFP are proficient in DNA binding and ATP hydrolysis. However, nucleoprotein complexes containing single-stranded DNA and RAD51A1-GFP or RAD51A2-GFP are significantly defective in binding to the second DNA molecule (secondary DNA binding), and consequently fail to catalyze homologous pairing. In contrast, RAD51A1-GFP and RAD51A2-GFP efficiently stimulated homologous pairing promoted by the meiosis-specific RAD51 isoform DMC1. These biochemical characteristics are well conserved in human RAD51-GFP. Therefore, GFP fused to the C terminus of RAD51 abolishes the homologous pairing activity of RAD51 by disrupting secondary DNA binding, but does not affect its DMC1-stimulating activity.

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“…However, the eukaryotic homologs differ from RecA in several notable respects. None of the eukaryotic enzymes appears to manifest the kinetic barrier to binding to duplex DNA (15), which in the case of RecA favors the loading of protein on single-stranded DNA; all hydrolyze ATP at a rate that is at least an order of magnitude lower than hydrolysis by RecA, and all promote recombination reactions much more slowly than RecA (10,11,12,14). Observations on human Rad51 showed that both phases of the recombination reaction, homologous pairing and strand exchange, are markedly slower than the corresponding phases of the RecA reactions (13).…”

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confidence: 99%

Human Rad51 Protein Can Form Homologous Joints in the Absence of Net Strand Exchange

Gupta

Folta‐Stogniew

Radding

1999

Journal of Biological Chemistry

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The eukaryotic homologs of RecA protein are central enzymes of recombination and repair, and notwithstanding a high degree of conservation they differ sufficiently from RecA to offer insights into mechanisms and biological roles. The yield of DNA strand exchange reactions driven by both Escherichia coli RecA protein and its human homolog HsRad51 protein was inversely related to the GC content of oligonucleotide substrates, but at any given GC composition, HsRad51 promoted less exchange than RecA. When 40% of bases were GC pairs, the rate constant for strand exchange by HsRad51 was unmeasurable, whereas the rate constants for homologous pairing were unaltered relative to more AT-rich DNA. The ability of HsRad51 to form joints in the absence of net strand exchange was confirmed by experiments in which heterologous blocks at both ends of linear duplex oligonucleotides produced joints that instantly dissociated upon deproteinization. These findings suggest that HsRad51 acting alone on human DNA in vivo is a pairing protein that cannot form extensive heteroduplex DNA.Human Rad51 protein (HsRad51) is a member of the universally distributed class of RecA proteins that play important roles in homologous recombination and recombinational repair (1). In prokaryotes, the RecA proteins play roles in recombination, postreplication repair, and the repair of double-strand breaks (2). In eukaryotes, members of this class play roles in meiotic recombination, double-strand break repair (3, 4), and possibly immunoglobulin switch recombination (5). In the mouse, the requirement of Rad51 for embryonic survival (6, 7) reveals a vital role that has not been found in prokaryotes and lower eukaryotes.RecA protein from Escherichia coli promotes a search for homology by a single strand of DNA, and initiates an exchange between that strand and homologous duplex DNA. To carry out those complicated interactions, RecA protein forms a helical nucleoprotein filament on single-stranded DNA; and Rad51 from Saccharomyces cerevisiae and Homo sapiens form nucleoprotein filaments that resemble the one formed by RecA (8,9). Once the nucleoprotein filament has been formed, RecA requires no cofactors other than ATP to promote a rapid search for homology and an extensive strand exchange. Yeast, frog, and human Rad51, as well as human Dmc1, a homolog that is specifically expressed in meiosis, are DNA-dependent ATPases that carry out homologous pairing and strand exchange reactions that resemble those catalyzed by RecA protein (10 -14). However, the eukaryotic homologs differ from RecA in several notable respects. None of the eukaryotic enzymes appears to manifest the kinetic barrier to binding to duplex DNA (15), which in the case of RecA favors the loading of protein on single-stranded DNA; all hydrolyze ATP at a rate that is at least an order of magnitude lower than hydrolysis by RecA, and all promote recombination reactions much more slowly than RecA (10,11,12,14). Observations on human Rad51 showed that both phases of the recombination reaction, homol...

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**Purification and characterization of XRad51.1 protein, Xenopus RAD51 homologue: recombinant XRad51.1 promotes strand exchange reaction**

Cited by 36 publications

References 26 publications

Recombination activities of HsDmc1 protein, the meiotic human homolog of RecA protein

Recombination activities of HsDmc1 protein, the meiotic human homolog of RecA protein

Green fluorescent protein fused to the C terminus of RAD51 specifically interferes with secondary DNA binding by the RAD51-ssDNA complex

Human Rad51 Protein Can Form Homologous Joints in the Absence of Net Strand Exchange

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