Functional analyses of the C-terminal half of the Saccharomyces cerevisiae Rad52 protein

Kagawa, Wataru; Arai, Naoto; Ichikawa, Yuichi; Saito, Kenkichi; Sugiyama, Shusei; Saotome, Mika; Shibata, Takehiko; Kurumizaka, Hitoshi

doi:10.1093/nar/gkt986

Cited by 13 publications

(19 citation statements)

References 38 publications

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“…For further clarification, we used anti-Rad51 antibody–protein A agarose to test whether E. coli DNA ligase co-precipitated with Rad51. In a positive control, Rad52, which is known to bind Rad51 at specific sites (12,44), was precipitated with Rad51 by the anti-Rad51 antibody-protein A agarose, but E. coli DNA ligase was not precipitated at all under these conditions (Figure 9). These results clearly indicated the absence of physical interactions between Rad51 and E. coli DNA ligase.…”

Section: Resultsmentioning

confidence: 99%

Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions

et al. 2016

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RecA-family recombinase-catalyzed ATP-dependent homologous joint formation is critical for homologous recombination, in which RecA or Rad51 binds first to single-stranded (ss)DNA and then interacts with double-stranded (ds)DNA. However, when RecA or Rad51 interacts with dsDNA before binding to ssDNA, the homologous joint-forming activity of RecA or Rad51 is quickly suppressed. We found that under these and adenosine diphosphate (ADP)-generating suppressive conditions for the recombinase activity, RecA or Rad51 at similar optimal concentrations enhances the DNA ligase-catalyzed dsDNA end-joining (DNA ligation) about 30- to 40-fold. The DNA ligation enhancement by RecA or Rad51 transforms most of the substrate DNA into multimers within 2–5 min, and for this enhancement, ADP is the common and best cofactor. Adenosine triphosphate (ATP) is effective for RecA, but not for Rad51. Rad51/RecA-enhanced DNA ligation depends on dsDNA-binding, as shown by a mutant, and is independent of physical interactions with the DNA ligase. These observations demonstrate the common and unique activities of RecA and Rad51 to juxtapose dsDNA-ends in preparation for covalent joining by a DNA ligase. This new in vitro function of Rad51 provides a simple explanation for our genetic observation that Rad51 plays a role in the fidelity of the end-joining of a reporter plasmid DNA, by yeast canonical non-homologous end-joining (NHEJ) in vivo.

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Section: Resultsmentioning

confidence: 99%

Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions

et al. 2016

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“…The C-terminal yRad51 binding region of yRad52 is required for mediator activity [ 19 , 26 ]. To investigate whether the same region is involved in the stimulation of DNA strand exchange by hRAD51, we constructed a derivative of yRad52 that lacked the C-terminal region (yRad52NM; Fig 1B and 1C ).…”

Section: Resultsmentioning

confidence: 99%

“…Based on this and other structural and biochemical evidence, we propose a model of yRad52-mediated annealing ( Fig 6 ). Crystallography has revealed that N-terminal half of hRAD52 adopts an undecameric ring structure in which the ssDNA binding groove runs around the outside rim of the ring (dashed line in Fig 6A ) [ 22 , 26 , 43 ]. Although the C-terminal region is not included in the crystal structure, we speculate that the C-terminal secondary DNA binding sites of yRad52 (green spheres in Fig 6 ) face to the primary ssDNA binding sites from outside of the ring.…”

Section: Discussionmentioning

confidence: 99%

“…The role of the C-terminal region of Rad52 seems to be considerably different in yeast and mammals. The C-terminal region (amino acids 327–504) of yRad52 is involved in protein-protein interaction with yRad51, and is required for mediator activity [ 18 , 19 , 25 , 26 ]. In contrast, attempts to detect recombination mediator activity of hRAD52 in vitro have been unsuccessful [ 4 , 27 ], although hRAD52 has the ability to bind hRAD51 in vitro .…”

Section: Introductionmentioning

confidence: 99%

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Roles of C-Terminal Region of Yeast and Human Rad52 in Rad51-Nucleoprotein Filament Formation and ssDNA Annealing

Khade

Sugiyama

2016

PLoS ONE

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Yeast Rad52 (yRad52) has two important functions at homologous DNA recombination (HR); annealing complementary single-strand DNA (ssDNA) molecules and recruiting Rad51 recombinase onto ssDNA (recombination mediator activity). Its human homolog (hRAD52) has a lesser role in HR, and apparently lacks mediator activity. Here we show that yRad52 can load human Rad51 (hRAD51) onto ssDNA complexed with yeast RPA in vitro. This is biochemically equivalent to mediator activity because it depends on the C-terminal Rad51-binding region of yRad52 and on functional Rad52-RPA interaction. It has been reported that the N-terminal two thirds of both yRad52 and hRAD52 is essential for binding to and annealing ssDNA. Although a second DNA binding region has been found in the C-terminal region of yRad52, its role in ssDNA annealing is not clear. In this paper, we also show that the C-terminal region of yRad52, but not of hRAD52, is involved in ssDNA annealing. This suggests that the second DNA binding site is required for the efficient ssDNA annealing by yRad52. We propose an updated model of Rad52-mediated ssDNA annealing.

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“…Yeast Rad52 (yRad52) is a key HR mediator that plays critical roles in Rad51 loading, DNA binding and single-stranded DNA (ssDNA) annealing during HR events [20]. Based on this mechanism, yRad52 was used to enhance gene targeting efficiency in mammalian cells to increase gene targeting by 37-fold via HR in Hela cells [21].…”

Section: Introductionmentioning

confidence: 99%

Enhancing Targeted Genomic DNA Editing in Chicken Cells Using the CRISPR/Cas9 System

Yang

Guo²,

et al. 2017

PLoS ONE

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The CRISPR/Cas9 system has enabled highly efficient genome targeted editing for various organisms. However, few studies have focused on CRISPR/Cas9 nuclease-mediated chicken genome editing compared with mammalian genomes. The current study combined CRISPR with yeast Rad52 (yRad52) to enhance targeted genomic DNA editing in chicken DF-1 cells. The efficiency of CRISPR/Cas9 nuclease-induced targeted mutations in the chicken genome was increased to 41.9% via the enrichment of the dual-reporter surrogate system. In addition, the combined effect of CRISPR nuclease and yRad52 dramatically increased the efficiency of the targeted substitution in the myostatin gene using 50-mer oligodeoxynucleotides (ssODN) as the donor DNA, resulting in a 36.7% editing efficiency after puromycin selection. Furthermore, based on the effect of yRad52, the frequency of exogenous gene integration in the chicken genome was more than 3-fold higher than that without yRad52. Collectively, these results suggest that ssODN is an ideal donor DNA for targeted substitution and that CRISPR/Cas9 combined with yRad52 significantly enhances chicken genome editing. These findings could be extensively applied in other organisms.

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Functional analyses of the C-terminal half of the Saccharomyces cerevisiae Rad52 protein

Cited by 13 publications

References 38 publications

Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions

Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions

Roles of C-Terminal Region of Yeast and Human Rad52 in Rad51-Nucleoprotein Filament Formation and ssDNA Annealing

Enhancing Targeted Genomic DNA Editing in Chicken Cells Using the CRISPR/Cas9 System

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