High Levels of Wild-Type BRCA2 Suppress Homologous Recombination

Magwood, Alissa C.; Mundia, Maureen M.; Baker, Mark D.

doi:10.1016/j.jmb.2012.05.007

Cited by 19 publications

(32 citation statements)

References 79 publications

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“…To gain further understanding of the requirement for BRCA2 (Brca2) in 39 extension, we exploited the stable mouse Brca2 knockdown cell line, 12-21 (Lee and Baker 2007). In 12-21, the process of 39 extension is compromised, but this can be augmented by overexpression of wild-type human BRCA2 supporting previous results for gene targeting in this cell line (Lee and Baker 2007;Magwood et al 2012). Furthermore, the inability of the human BRCA2 variant bearing an in-frame deletion of the Rad51-binding BRC repeats 1-8 (BRCA2 Δ1-8 ) to alleviate the deficit in 39 extension in the recipient 12-21 cell line supports an important role for BRCA2 BRC repeats in loading/stabilizing Rad51 during nucleoprotein filament formation in the early steps of homologous recombination in vivo.…”

Section: Discussionsupporting

confidence: 55%

“…Our previous studies support a role for BRCA2 (Brca2) in regulating 39 extension in vivo: moderate expression of human BRCA2 in the igm482 hybridoma (which expresses endogenous mouse Brca2) increased 39 extension and HR, while high BRCA2 overexpression suppressed 39 extension and HR (Magwood et al 2012). To gain further understanding of the requirement for BRCA2 (Brca2) in 39 extension, we exploited the stable mouse Brca2 knockdown cell line, 12-21 (Lee and Baker 2007).…”

Section: Discussionmentioning

confidence: 77%

“…The Brca2-depleted 12-21 cell line is an igm482 derivative that stably expresses anti-mouse Brca2 small interfering RNA (siRNA) (Lee and Baker 2007), while 12-21 derivatives that express wild-type human BRCA2 (in BAC clone RP11-777I19; Sharan et al 2004) were established as described (Magwood et al 2012). All cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 13% bovine calf serum, penicillin/streptomycin, and 2-mercaptoethanol as described (Köhler and Shulman 1980;Köhler et al 1982).…”

Section: Cell Lines and Plasmidsmentioning

confidence: 99%

“…Western blot and immunoprecipitation analyses were performed according to Magwood et al (2012) with the exception that rabbit IgG serum (ThermoScientific) was used as a nonspecific antisera in immunoprecipitation. The following primary antibodies were used: mouse monoclonal anti-human Rad51 (14B4, Abcam), mouse monoclonal anti-FLAG (M2, Sigma-Aldrich), mouse monoclonal anti-b-actin (AC-15, SigmaAldrich), mouse monoclonal anti-human histone H1 (A-E4, Santa Cruz Biotechnology), rabbit polyclonal anti-human caspase-3 (SA-320, Enzo Life Sciences), and rabbit polyclonal anti-human BRCA2 (Ab27976, Abcam).…”

Section: Protein Analysismentioning

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: Discussionsupporting

confidence: 55%

Section: Discussionmentioning

confidence: 77%

Section: Cell Lines and Plasmidsmentioning

confidence: 99%

Section: Protein Analysismentioning

confidence: 99%

Section: Brca2 Is Required For 39 Extensionmentioning

confidence: 99%

See 3 more Smart Citations

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

et al. 2014

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

et al. 2013

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The dichotomous effects of caffeine on homologous recombination in mammalian cells

et al. 2020

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This study was initiated to examine the effects of caffeine on the DNA damage response (DDR) and homologous recombination (HR). An initial 2 h exposure to 5 mM caffeine slowed a fraction of the cells in G1, but thereafter, continued caffeine exposure permitted this cell fraction to progress through the cycle until they eventually stalled at G2/M and underwent apoptosis. This prolonged caffeine exposure also induced a strong DDR along with subsequent activation of wild-type p53 protein. An unexpected observation was the caffeine-induced depletion of Rad51 (and Brca2) proteins. Consequently, caffeine-treated cells were expected to be inefficient in HR. However, a dichotomy in the HR response of cells to caffeine treatment was revealed. Caffeine treatment rendered cells significantly better at performing the nascent DNA synthesis that accompanies the early strand invasion steps of HR. Conversely, the increase in nascent DNA synthesis did not translate into a higher level of gene targeting events. Levels of Rad51 appear to be irrelevant. Thus, prolonged caffeine exposure stalls the cell cycle, induces a p53-mediated apoptotic response and a down-regulation of critical HR proteins, and stimulates early steps of HR, but not the formation of complete repair products. INTRODUCTIONThe DNA damage response (DDR) is a complex signalling pathway consisting of sensors, transducers and effectors that evolved to detect DNA damage, halt the cell cycle and co-ordinate an adaptive response (1). In mammalian cells, the protein kinases ATM (ataxia-telangiectasia mutated) and ATR (ATM-and Rad3-related), both members of the phosphatidylinositol 3kinase-related kinase (PIKK) superfamily of protein kinases play a key role in DDR signalling (2). The CHK1 and CHK2 protein kinases are two of the best studied ATM/ATR targets and in association with ATM and ATR, reduce cyclin-dependent kinase (CDK) activity by mechanisms that include activation of the p53 transcription factor (3). CDK inhibition reduces or halts cell cycle progression at either of the G1-S, intra-S or G2-M cell cycle checkpoint boundaries (3). Cell cycle arrest normally permits repair of DNA damage prior to DNA replication or mitosis through ATM-and ATR-catalyzed induction of DNA repair protein transcription or posttranscriptional activation, recruitment of DNA repair factors to the damaged site and/or their

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High Levels of Wild-Type BRCA2 Suppress Homologous Recombination

Cited by 19 publications

References 79 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

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

The dichotomous effects of caffeine on homologous recombination in mammalian cells

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