An RNF168 fragment defective for focal accumulation at DNA damage is proficient for inhibition of homologous recombination in BRCA1 deficient cells

Muñoz, Meilen C.; Yanez, Diana A.; Stark, Jeremy M.

doi:10.1093/nar/gku421

Cited by 28 publications

(31 citation statements)

References 55 publications

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“…We hypothesized that the predicted 5′ overhang structure could be processed to yield a recombination favourable 3′ overhang. If this were the case, GC would be sensitive to an ectopically expressed 3′–5′ exonuclease such as TREX2 known to process 3′ overhangs282930. Indeed, co-expression of TREX2 and D10A Cas9 resulted in a strong reduction in GC frequency, indicating that a 3′ overhang is part of the life cycle of a 5′ overhang undergoing GC independent of the gRNA pair (Fig.…”

Section: Resultsmentioning

confidence: 96%

Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus

Bothmer¹,

Phadke²,

Barrera³

et al. 2017

Nat Commun

170

184

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The CRISPR–Cas9 system provides a versatile toolkit for genome engineering that can introduce various DNA lesions at specific genomic locations. However, a better understanding of the nature of these lesions and the repair pathways engaged is critical to realizing the full potential of this technology. Here we characterize the different lesions arising from each Cas9 variant and the resulting repair pathway engagement. We demonstrate that the presence and polarity of the overhang structure is a critical determinant of double-strand break repair pathway choice. Similarly, single nicks deriving from different Cas9 variants differentially activate repair: D10A but not N863A-induced nicks are repaired by homologous recombination. Finally, we demonstrate that homologous recombination is required for repairing lesions using double-stranded, but not single-stranded DNA as a template. This detailed characterization of repair pathway choice in response to CRISPR–Cas9 enables a more deterministic approach for designing research and therapeutic genome engineering strategies.

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Section: Resultsmentioning

confidence: 96%

Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus

Bothmer¹,

Phadke²,

Barrera³

et al. 2017

Nat Commun

170

184

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“…This reporter assay typically involves using I-SceI to induce the DSB, but, to remain consistent across assays, we used an sgRNA to target Cas9 to the I-SceI site in DR-GFP (Supplemental Fig. S2), as described previously (Munoz et al 2014). We found that expression vectors for 3xf-NLS-BRC3 and RAD51-K133R, compared with control empty vector (EV), each caused a substantial decrease in HDR ( Fig.…”

Section: Rmds At Dsb/repeat Distances ≥33 Kb Are Inhibited By Rad51 mentioning

confidence: 84%

Repeat-mediated deletions can be induced by a chromosomal break far from a repeat, but multiple pathways suppress such rearrangements

2018

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Chromosomal deletion rearrangements mediated by repetitive elements often involve repeats separated by several kilobases and sequences that are divergent. While such rearrangements are likely induced by DNA double-strand breaks (DSBs), it has been unclear how the proximity of DSBs relative to repeat sequences affects the frequency of such events. We generated a reporter assay in mouse cells for a deletion rearrangement involving repeats separated by 0.4 Mb. We induced this repeat-mediated deletion (RMD) rearrangement with two DSBs: the 5' DSB that is just downstream from the first repeat and the 3' DSB that is varying distances upstream of the second repeat. Strikingly, we found that increasing the 3' DSB/repeat distance from 3.3 kb to 28.4 kb causes only a modest decrease in rearrangement frequency. We also found that RMDs are suppressed by KU70 and RAD51 and promoted by RAD52, CtIP, and BRCA1. In addition, we found that 1%-3% sequence divergence substantially suppresses these rearrangements in a manner dependent on the mismatch repair factor MSH2, which is dominant over the suppressive role of KU70. We suggest that a DSB far from a repeat can stimulate repeat-mediated rearrangements, but multiple pathways suppress these events.

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“…In addition to the classic role of RNF8 described above, other effects of RNF8 have been reported. Studies have confirmed that RNF8 depletion partially suppresses HR defects caused by BRCA1 depletion, and the degree of HR suppression due to RNF8 depletion is significantly less than that due to RNF168 depletion . Moreover, scientists revealed a newly discovered non‐catalytic role of RNF8 in unfolding higher‐order chromatin structures .…”

Section: Introductionmentioning

confidence: 95%

“…Studies have confirmed that RNF8 depletion partially suppresses HR defects caused by BRCA1 depletion, and the degree of HR suppression due to RNF8 depletion is significantly less than that due to RNF168 depletion. 15 Moreover, scientists revealed a newly discovered non-catalytic role of RNF8 in unfolding higher-order chromatin structures. 16 Furthermore, RNF8 has been shown to promote cellular resistance to replication stress and is important for meiosis.…”

mentioning

confidence: 99%

RNF8 promotes efficient DSB repair by inhibiting the pro‐apoptotic activity of p53 through regulating the function of Tip60

et al. 2020

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Objectives RING finger protein 8 (RNF8) is an E3 ligase that plays an essential role in DSB repair. p53 is a well‐established tumour suppressor and cellular gatekeeper of genome stability. This study aimed at investigating the functional correlations between RNF8 and p53 in DSB damage repair. Materials and methods In this article, wild‐type, knockout and shRNA‐depleted HCT116 and U2OS cells were stressed, and the roles of RNF8 and p53 were examined. RT‐PCR and Western blot were utilized to investigate the expression of related genes in damaged cells. Cell proliferation, apoptosis and neutral cell comet assays were applied to determine the effects of DSB damage on differently treated cells. DR‐GFP, EJ5‐GFP and LacI‐LacO targeting systems, flow cytometry, mass spectrometry, IP, IF, GST pull‐down assay were used to explore the molecular mechanism of RNF8 and p53 in DSB damage repair. Results We found that RNF8 knockdown increased cellular sensitivity to DSB damage and decreased cell proliferation, which was correlated with high expression of the p53 gene. RNF8 improved the efficiency of DSB repair by inhibiting the pro‐apoptotic function of p53. We also found that RNF8 restrains cell apoptosis by inhibiting over‐activation of ATM and subsequently reducing p53 acetylation at K120 through regulating Tip60. Conclusions Taken together, these findings suggested that RNF8 promotes efficient DSB repair by inhibiting the pro‐apoptotic activity of p53 through regulating the function of Tip60.

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An RNF168 fragment defective for focal accumulation at DNA damage is proficient for inhibition of homologous recombination in BRCA1 deficient cells

Cited by 28 publications

References 55 publications

Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus

Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus

Repeat-mediated deletions can be induced by a chromosomal break far from a repeat, but multiple pathways suppress such rearrangements

RNF8 promotes efficient DSB repair by inhibiting the pro‐apoptotic activity of p53 through regulating the function of Tip60

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