Endogenous levels of Rad51 and Brca2 are required for homologous recombination and regulated by homeostatic re-balancing

Magwood, Alissa C.; Malysewich, Michael J.; Cealic, Iulia; Mundia, Maureen M.; Knapp, Jennifer R.; Baker, Mark D.

doi:10.1016/j.dnarep.2013.10.006

Cited by 22 publications

(52 citation statements)

References 65 publications

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“…However, significantly lower levels of FLAG-Rad51 protein are observed in cell lines expressing the F86E/A89E double mutant (#2, lane 7; #14, lane 8), suggesting that this Rad51 variant is more toxic to the hybridoma cells. As reported previously (Magwood et al 2013), slight variation in the endogenous Rad51 levels in the various transformants is due to multiple Rad51 translational start sites that result in a transgene contribution to the level of "endogenous" Rad51.…”

Section: Role Of Rad51 Polymerization In Association Of Rad51 and Brca2mentioning

confidence: 75%

“…Previously, we showed that cell lines siRNA-depleted for endogenous Rad51 are crippled for 39 extension (Magwood et al 2013). Therefore, in this study, it was of interest to further examine how Rad51 concentration influences the Figure 5 Cellular distribution of Rad51 polymerization mutants.…”

Section: Discussionmentioning

confidence: 99%

“…To determine the requirement for Brca2 in 39 extension, we utilized mouse hybridoma cell line 12-21 that is 75% siRNA depleted for mouse Brca2 (Lee et al 2009) along with 12-21 derivatives that express either ectopic wild-type human BRCA2 or a human BRCA2 mutant bearing an in-frame deletion of BRC repeats 1-8 (Magwood et al 2012(Magwood et al , 2013. As shown in Figure 7, at 6 hr, the frequency of the 39 extension/vector backbone is significantly reduced in the mouse Brca2 knockdown cell line 12-21 compared to control igm482 cells.…”

Section: Brca2 Is Required For 39 Extensionmentioning

confidence: 99%

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Nascent DNA Synthesis During Homologous Recombination Is Synergistically Promoted by the Rad51 Recombinase and DNA Homology

et al. 2014

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In this study, we exploited a plasmid-based assay that detects the new DNA synthesis (39 extension) that accompanies Rad51-mediated homology searching and strand invasion steps of homologous recombination to investigate the interplay between Rad51 concentration and homology length. Mouse hybridoma cells that express endogenous levels of Rad51 display an approximate linear increase in the frequency of 39 extension for homology lengths of 500 bp to 2 kb. At values below 500 bp, the frequency of 39 extension declines markedly, suggesting that this might represent the minimal efficient processing segment for 39 extension. Overexpression of wild-type Rad51 stimulated the frequency of 39 extension by 3-fold for homology lengths ,900 bp, but when homology was .2 kb, 39 extension frequency increased by as much as 10-fold. Excess wild-type Rad51 did not increase the average 39 extension tract length. Analysis of cell lines expressing N-terminally FLAG-tagged Rad51 polymerization mutants F86E, A89E, or F86E/A89E established that the 39 extension process requires Rad51 polymerization activity. Mouse hybridoma cells that have reduced Brca2 (Breast cancer susceptibility 2) due to stable expression of small interfering RNA show a significant reduction in 39 extension efficiency; expression of wild-type human BRCA2, but not a BRCA2 variant devoid of BRC repeats 1-8, rescues the 39 extension defect in these cells. Our results suggest that increased Rad51 concentration and homology length interact synergistically to promote 39 extension, presumably as a result of enhanced Brca2-mediated Rad51 polymerization. Homologous recombination is a complex, multi-step process (reviewed in Brugmans et al. 2007). In eukaryotes, the central step in the repair of a DNA DSB involves the binding of multiple monomers of the RAD51 protein (a functional homolog of the Escherichia coli RecA recombinase) to 39-ending single-stranded DNA overhangs created by nucleolytic resection (Van Den Bosch et al. 2003). The resulting RAD51 nucleoprotein filament promotes formation of a joint molecule between the processed broken DNA and the homologous repair template by the orchestrated steps of homology searching and DNA strand invasion and exchange. Joint molecule formation is followed by new DNA synthesis, which replaces nucleotides lost through DSB formation and 39-end resection (Pâques and Haber 1999;Symington 2002;Li and Heyer 2008). Depending on the subpathway of HR, subsequent steps may involve unwinding (dissolution) of the DNA strand containing the newly synthesized DNA and ligation to the processed second end or the formation of a stably joined Holliday junction intermediate that can be processed further by structure-specific endonucleases yielding recombinant products (Li and Heyer 2008).At the heart of the HR process is the Rad51 (or RecA) nucleoprotein filament, whose formation requires the initial association of four to five monomers with 39-ending singlestranded DNA in the step known as nucleation (Galletto

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Section: Role Of Rad51 Polymerization In Association Of Rad51 and Brca2mentioning

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Section: Brca2 Is Required For 39 Extensionmentioning

confidence: 99%

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Nascent DNA Synthesis During Homologous Recombination Is Synergistically Promoted by the Rad51 Recombinase and DNA Homology

et al. 2014

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“…2a, b, Supplementary Fig. 4C) 24, 37 . Of note, the reduction in RAD51 protein by itself did not account for impaired RAD51 accumulation at DSBs, as DSB-associated RAD51 levels were normalized to overall nuclear RAD51 abundance and, thus, reflect a bona-fide RAD51 recruitment defect (Fig.…”

Section: Resultsmentioning

confidence: 99%

Ubiquitin-specific protease 21 stabilizes BRCA2 to control DNA repair and tumor growth

et al. 2017

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Tumor growth relies on efficient DNA repair to mitigate the detrimental impact of DNA damage associated with excessive cell division. Modulating repair factor function, thus, provides a promising strategy to manipulate malignant growth. Here, we identify the ubiquitin-specific protease USP21 as a positive regulator of BRCA2, a key mediator of DNA repair by homologous recombination. USP21 interacts with, deubiquitinates and stabilizes BRCA2 to promote efficient RAD51 loading at DNA double-strand breaks. As a result, depletion of USP21 decreases homologous recombination efficiency, causes an increase in DNA damage load and impairs tumor cell survival. Importantly, BRCA2 overexpression partially restores the USP21-associated survival defect. Moreover, we show that USP21 is overexpressed in hepatocellular carcinoma, where it promotes BRCA2 stability and inversely correlates with patient survival. Together, our findings identify deubiquitination as a means to regulate BRCA2 function and point to USP21 as a potential therapeutic target in BRCA2-proficient tumors.

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“…Proteins involved in HR include Rad51, RPA, XRCC2, BRCA1, BRCA2, etc. [1][2][3]. By contrast, NHEJ is the most straightforward way to repair a DSB, as the broken ends are simply rejoined regardless of the genetic sequences at the break.…”

Section: Introductionmentioning

confidence: 99%

Efficient ligase 3-dependent microhomology-mediated end joining repair of DNA double-strand breaks in zebrafish embryos

Zhang

Wang

et al. 2015

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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Endogenous levels of Rad51 and Brca2 are required for homologous recombination and regulated by homeostatic re-balancing

Cited by 22 publications

References 65 publications

Nascent DNA Synthesis During Homologous Recombination Is Synergistically Promoted by the Rad51 Recombinase and DNA Homology

Nascent DNA Synthesis During Homologous Recombination Is Synergistically Promoted by the Rad51 Recombinase and DNA Homology

Ubiquitin-specific protease 21 stabilizes BRCA2 to control DNA repair and tumor growth

Efficient ligase 3-dependent microhomology-mediated end joining repair of DNA double-strand breaks in zebrafish embryos

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