Caffeine inhibits gene conversion by displacing Rad51 from ssDNA

Tsabar, Michael; Mason, Jennifer M.; Chan, Yuen‐Ling; Bishop, Douglas K.; Haber, James E.

doi:10.1093/nar/gkv525

Cited by 17 publications

(22 citation statements)

References 88 publications

(107 reference statements)

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“…To test directly if Rad51-GFP was bound to the DNA around the DSB, we performed chromatin immunoprecipitation using an antibody recognizing Rad51 to assay Rad51-GFP accumulation 5 kb from the DSB induced in the MATa locus in a derivative of strain JKM179, lacking donor sequences, as described previously (28). As shown in Figure 1G, Rad51-GFP binding 5 kb from the unrepaired DSB end increased steadily over 6 h. Rad51-GFP binding appears similar in its kinetics to wild type Rad51, as measured previously (19, 28). Therefore, Rad51-GFP effectively binds to resected DNA around a DSB and thus shows promise for further live cell studies.…”

Section: Resultssupporting

confidence: 84%

“…Previously, a Rad51-GFP fusion was characterized in Arabidopsis, where it proved to be defective in mitotic DSB repair, but competent in meiosis (17). 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).…”

Section: Introductionmentioning

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: Resultssupporting

confidence: 84%

Section: Introductionmentioning

confidence: 99%

Live cell monitoring of double strand breaks inS. cerevisiae

Waerman

Lee

Tsabar

et al. 2018

Preprint

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“…6e ). Previous results showed that caffeine treatment affected HR independently of its inhibition of checkpoint kinases, which are essential for the HR pathway 30 , 31 . Accordingly, the formation of both Rad51 and Rad54 foci were significantly decreased following caffeine treatment, which is expected to impair HR in ES cells.…”

Section: Resultsmentioning

confidence: 98%

The Homologous Recombination Machinery Orchestrates Post-replication DNA Repair During Self-renewal of Mouse Embryonic Stem Cells

Choi

Yoon

Park

et al. 2017

Sci Rep

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Embryonic stem (ES) cells require homologous recombination (HR) to cope with genomic instability caused during self-renewal. Here, we report expression dynamics and localization of endogenous HR factors in DNA break repair of ES cells. In addition, we analyzed gene expression patterns of HR-related factors at the transcript level with RNA-sequencing experiments. We showed that ES cells constitutively expressed diverse HR proteins throughout the cell cycle and that HR protein expression was not significantly changed even in the DNA damaging conditions. We further analyzed that depleting Rad51 resulted in the accumulation of larger single-stranded DNA (ssDNA) gaps, but did not perturb DNA replication, indicating that ES cells were able to enter the G2-phase in the presence of unrepaired DNA gaps, consistent with the possibility that post-replication repair helps avoid stalling at the G2/M checkpoint. Interestingly, caffeine treatment inhibited the formation of Rad51 or Rad54 foci, but not the formation of γH2AX and Exo1 foci, which led to incomplete HR in ssDNA, thus increasing DNA damage sensitivity. Our results suggested that ES cells possess conserved HR-promoting machinery to ensure effective recruitment of the HR proteins to DNA breaks, thereby driving proper chromosome duplication and cell cycle progression in ES cells.

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“…Therefore, we performed experiments in the presence of chemical inhibitors: KU-55933 specific for ATM [58], VE-821 specific for ATR [59], wortmannin that primarily affects DNA-PKcs [60], and the Chk1 inhibitor UCN-01 [61] at the concentrations we previously found to be effective in mouse ES cells [62]. We also tested the effect of caffeine, which is widely used as a broadly specific ATM/ATR/DNA-PKcs inhibitor, but which we found to lack this activity in ES cells, and to strongly suppress gene targeting by HR [62][63][64]. Inhibition of ATM and ATR reduced S-RI efficiency (S2E and S2F Fig), and the combination of the two inhibitors had an additive effect, which was even more pronounced in DnaPKcs -/cells (S2E and S2F Fig).…”

Section: S-ri Requires γH2ax But Not Hr or Nhejmentioning

confidence: 99%

Low dose ionizing radiation strongly stimulates insertional mutagenesis in a γH2AX dependent manner

et al. 2020

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Extrachromosomal DNA can integrate into the genome with no sequence specificity producing an insertional mutation. This process, which is referred to as random integration (RI), requires a double stranded break (DSB) in the genome. Inducing DSBs by various means, including ionizing radiation, increases the frequency of integration. Here we report that nonlethal physiologically relevant doses of ionizing radiation (10-100 mGy), within the range produced by medical imaging equipment, stimulate RI of transfected and viral episomal DNA in human and mouse cells with an extremely high efficiency. Genetic analysis of the stimulated RI (S-RI) revealed that it is distinct from the background RI, requires histone H2AX S139 phosphorylation (γH2AX) and is not reduced by DNA polymerase θ (Polq) inactivation. S-RI efficiency was unaffected by the main DSB repair pathway (homologous recombination and non-homologous end joining) disruptions, but double deficiency in MDC1 and 53BP1 phenocopies γH2AX inactivation. The robust responsiveness of S-RI to physiological amounts of DSBs can be exploited for extremely sensitive, macroscopic and direct detection of DSB-induced mutations, and warrants further exploration in vivo to determine if the phenomenon has implications for radiation risk assessment.

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Caffeine inhibits gene conversion by displacing Rad51 from ssDNA

Cited by 17 publications

References 88 publications

Live cell monitoring of double strand breaks inS. cerevisiae

Live cell monitoring of double strand breaks inS. cerevisiae

The Homologous Recombination Machinery Orchestrates Post-replication DNA Repair During Self-renewal of Mouse Embryonic Stem Cells

Low dose ionizing radiation strongly stimulates insertional mutagenesis in a γH2AX dependent manner

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