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

Kobayashi, Wataru; Sekine, Satoshi; Machida, Shuichi; Kurumizaka, Hitoshi

doi:10.1266/ggs.89.169

Cited by 18 publications

(32 citation statements)

References 61 publications

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“…This phenotype resembles the “site II” mutation of Saccharomyces cerevisiae Rad51, which can bind ssDNA but is unable to bind dsDNA and thus fails to complete strand invasion and DSB repair in mitotic cells (18,19). Similar results were obtained using a human isoform of Rad51-GFP in vitro (20). In fission yeast, scRad51’s homolog Rhp51 when fused with CFP proved to be UV sensitive and incapable of carrying our repair on its own, but this defect was complimented by expression of wild type Rhp51 (21).…”

Section: Introductionsupporting

confidence: 83%

“…While our Rad51-GFP construct is not able, by itself, to repair DSBs by homologous recombination, it is not dominant negative and supports recombination in meiosis. Biochemical work on a human isoform of Rad51 fused to GFP determined that the fluorescent tag prevented Rad51 from engaging in the pairing of homologous sequences by inhibiting Rad51’s secondary DNA binding (20). We envision the same to be true of our Rad51-GFP construct because our ChIP experiments and microscopy suggest that Rad51-GFP can efficiently bind to ssDNA and form a filament, its first step in homologous recombination.…”

Section: Discussionmentioning

confidence: 99%

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Live cell monitoring of double strand breaks inS. cerevisiae

Waerman

Lee

Tsabar

et al. 2018

Preprint

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We have used two different live-cell fluorescent protein markers to monitor the formation and localization of double-strand breaks (DSBs) in budding yeast. Using GFP derivatives of the Rad51 recombination protein or the Ddc2 checkpoint protein, we find that cells with three site-specific DSBs, on different chromosomes, usually display 2 or 3 foci that coalesce and dissociate. Rad51-GFP, by itself, is unable to repair DSBs by homologous recombination in mitotic cells, but is able to form foci and allow repair when heterozygous with a wild type Rad51 protein. The kinetics of disappearance of Rad51-GFP foci parallels the completion of DSB repair. However, in meiosis, Rad51-GFP is proficient when homozygous. Using Ddc2-GFP, we conclude that co-localization of foci following 3 DSBs does not represent formation of a homologous recombination "repair center," as the same distribution of Ddc2-GFP foci was found in the presence or absence of the Rad52 protein. The maintenance of separate DSB foci and much of their dynamics depend on functional microtubules, as addition of nocodazole resulted in a greater population of cells displaying a single focus.Author SummaryDouble strand breaks (DSBs) pose the greatest threat to the fidelity of an organism’s genome. While much work has been done on the mechanisms of DSB repair, the arrangement and interaction of multiple DSBs within a single cell remain unclear. Using two live-cell fluorescent DSB markers, we show that cells with 3 site-specific DSBs usually form 2 or 3 foci what can coalesce into fewer foci but also dissociate. The aggregation of DSBs into a single focus does not depend on the Rad52 recombination protein, suggesting that there is no “repair center” for homologous recombination. DSB foci are highly dynamic and their dynamic nature is dependent on microtubules.

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Section: Introductionsupporting

confidence: 83%

Section: Discussionmentioning

confidence: 99%

Live cell monitoring of double strand breaks inS. cerevisiae

Waerman

Lee

Tsabar

et al. 2018

Preprint

View full text Add to dashboard Cite

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“…; Kobayashi et al . ). A recent analysis of the Rad51 knockdown in mouse testis showed the depletion of primary spermatocytes and the reduction in crossover formation (Dai et al .…”

Section: Introductionmentioning

confidence: 97%

“…Dmc1À/À mice grow normally, but are infertile due to failure of chromosome synapsis (Pittman et al 1998;Yoshida et al 1998). In meiosis, RAD51 also acts as an accessory factor to promote DMC1-mediated strand invasion (Cloud et al 2012;Da Ines et al 2013;Kobayashi et al 2014). A recent analysis of the Rad51 knockdown in mouse testis showed the depletion of primary spermatocytes and the reduction in crossover formation (Dai et al 2017).…”

Section: Introductionmentioning

confidence: 99%

SYCP3 regulates strand invasion activities of RAD51 and DMC1

et al. 2017

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The synaptonemal complex is a higher-ordered proteinaceous architecture formed between homologous chromosomes. SYCP3 is a major component of the lateral/axial elements in the synaptonemal complex and is essential for meiotic recombination. Previous genetic studies showed that SYCP3 functions in meiotic homologous recombination biased to interhomologous chromosomes, by regulating the strand invasion activities of the RAD51 and DMC1 recombinases. However, the mechanism by which SYCP3 regulates RAD51- and DMC1-mediated strand invasion remains elusive. In this study, we found that SYCP3 significantly suppresses the RAD51-mediated, but not the DMC1-mediated, strand invasion reaction by competing with HOP2-MND1, which is an activator for both RAD51 and DMC1. A SYCP3 mutant with defective RAD51 binding does not inhibit the RAD51-mediated homologous recombination in human cells. Therefore, SYCP3 may promote the DMC1-driven homologous recombination by attenuating the RAD51 activity during meiosis.

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“…Inactivation of the secondary DNA binding site of RAD51 in rad51-II3A mutant yeast blocks its ability to catalyse recombination but does not affect fertility [ 30 ]. This is also seen upon expression of the dominant-negative RAD51-GFP fusion protein in Arabidopsis [ 31 ], which also lacks secondary DNA binding and strand-invasion activity [ 52 ]. In contrast to the effect of absence of RAD51, these mutant RAD51 proteins are unable to catalyse invasion of the template DNA duplex and are defective in mitotic DSB repair, but remain able to support the activity of DMC1 in meiosis [ 30 , 31 , 52 ].…”

Section: Introductionmentioning

confidence: 99%

Analysis of the impact of the absence of RAD51 strand exchange activity in Arabidopsis meiosis

et al. 2017

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The ploidy of eukaryote gametes must be halved to avoid doubling of numbers of chromosomes with each generation and this is carried out by meiosis, a specialized cell division in which a single chromosomal replication phase is followed by two successive nuclear divisions. With some exceptions, programmed recombination ensures the proper pairing and distribution of homologous pairs of chromosomes in meiosis and recombination defects thus lead to sterility. Two highly related recombinases are required to catalyse the key strand-invasion step of meiotic recombination and it is the meiosis-specific DMC1 which is generally believed to catalyse the essential non-sister chromatid crossing-over, with RAD51 catalysing sister-chromatid and non-cross-over events. Recent work in yeast and plants has however shown that in the absence of RAD51 strand-exchange activity, DMC1 is able to repair all meiotic DNA breaks and surprisingly, that this does not appear to affect numbers of meiotic cross-overs. In this work we confirm and extend this conclusion. Given that more than 95% of meiotic homologous recombination in Arabidopsis does not result in inter-homologue crossovers, Arabidopsis is a particularly sensitive model for testing the relative importance of the two proteins—even minor effects on the non-crossover event population should produce detectable effects on crossing-over. Although the presence of RAD51 protein provides essential support for the action of DMC1, our results show no significant effect of the absence of RAD51 strand-exchange activity on meiotic crossing-over rates or patterns in different chromosomal regions or across the whole genome of Arabidopsis, strongly supporting the argument that DMC1 catalyses repair of all meiotic DNA breaks, not only non-sister cross-overs.

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

Cited by 18 publications

References 61 publications

Live cell monitoring of double strand breaks inS. cerevisiae

Live cell monitoring of double strand breaks inS. cerevisiae

SYCP3 regulates strand invasion activities of RAD51 and DMC1

Analysis of the impact of the absence of RAD51 strand exchange activity in Arabidopsis meiosis

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