ROS-induced R loops trigger a transcription-coupled but BRCA1/2-independent homologous recombination pathway through CSB

Teng, Yaqun; Yadav, Tribhuwan; Duan, Meihan; Tan, Jun; Xiang, Yufei; Gao, Boya; Xu, Jianquan; Liang, Zhuobin; Liu, Yang; Nakajima, Satoshi; Shi, Yi; Levine, Arthur S.; Zou, Lee; Lan, Li

doi:10.1038/s41467-018-06586-3

Cited by 134 publications

(146 citation statements)

References 41 publications

(58 reference statements)

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“…DDX5 or PRMT5 knockdown (Fig A) led to a significant increase in R‐loop specifically at the TA‐KR marked locus, while the level of R‐loops was similar to the control at the TetR‐KR locus (Fig B and C). The signal we observe is similar to published studies (Teng et al , ). Under the conditions used (the staining involves a heating step on a 95°C heating block for 20 min to expose the antigen and blocking with 5% BSA), the S9.6 focus is more apparent than the typical nucleolar staining observed with standard S9.6 staining protocols.…”

Section: Resultssupporting

confidence: 93%

“…KR generates reactive oxygen species (ROS) through the excited chromophore and induces DNA damage and transcriptional activation at the genome‐integrated tet response element (TRE) locus in the U2OS TRE cells. Elevated R‐loop at the TRE locus over background is visualized by S9.6 immunofluorescence (Teng et al , ; Liang et al , ). DDX5 or PRMT5 knockdown (Fig A) led to a significant increase in R‐loop specifically at the TA‐KR marked locus, while the level of R‐loops was similar to the control at the TetR‐KR locus (Fig B and C).…”

Section: Resultsmentioning

confidence: 99%

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Arginine methylation of the DDX 5 helicase RGG / RG motif by PRMT 5 regulates resolution of RNA:DNA hybrids

et al. 2019

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Aberrant transcription‐associated RNA : DNA hybrid (R‐loop) formation often causes catastrophic conflicts during replication, resulting in DNA double‐strand breaks and genomic instability. Preventing such conflicts requires hybrid dissolution by helicases and/or RN ase H. Little is known about how such helicases are regulated. Herein, we identify DDX 5, an RGG / RG motif‐containing DEAD ‐box family RNA helicase, as crucial player in R‐loop resolution. In vitro , recombinant DDX 5 resolves R‐loops in an ATP ‐dependent manner, leading to R‐loop degradation by the XRN 2 exoribonuclease. DDX 5‐deficient cells accumulate R‐loops at loci with propensity to form such structures based on RNA : DNA immunoprecipitation ( DRIP )‐ qPCR , causing spontaneous DNA double‐strand breaks and hypersensitivity to replication stress. DDX 5 associates with XRN 2 and resolves R‐loops at transcriptional termination regions downstream of poly(A) sites, to facilitate RNA polymerase II release associated with transcriptional termination. Protein arginine methyltransferase 5 ( PRMT 5) binds and methylates DDX 5 at its RGG / RG motif. This motif is required for DDX 5 interaction with XRN 2 and repression of cellular R‐loops, but not essential for DDX 5 helicase enzymatic activity. PRMT 5‐deficient cells accumulate R‐loops, resulting in increased formation of γH2 AX foci. Our findings exemplify a mechanism by which an RNA helicase is modulated by arginine methylation to resolve R‐loops, and its potential role in regulating transcription.

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Section: Resultssupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Arginine methylation of the DDX 5 helicase RGG / RG motif by PRMT 5 regulates resolution of RNA:DNA hybrids

et al. 2019

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“…There is also evidence that R-loops may recruit factors that participate in DSB repair. Cockayne syndrome protein B (CSB) is likely such a factor (84). CSB participates in DSB repair by recruiting Rad52, which then recruits Rad51, a protein involved in the strand invasion step of homologous recombination (Fig.…”

Section: Jbc Reviews: R-loop Benefits and Risksmentioning

confidence: 99%

“…There are still many unanswered questions concerning the role of R-loop formation in double-strand break repair. Research covering the subject describes processes such as transcription-associated homologous recombination repair and transcription-coupled homologous recombination (84,85), but it is unclear whether these processes are independent or in some way linked-perhaps even to the point of being the same phenomenon.…”

Section: Jbc Reviews: R-loop Benefits and Risksmentioning

confidence: 99%

The balancing act of R-loop biology: The good, the bad, and the ugly

2019

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An R-loop is a three-stranded nucleic acid structure that consists of a DNA:RNA hybrid and a displaced strand of DNA. R-loops occur frequently in genomes and have significant physiological importance. They play vital roles in regulating gene expression, DNA replication, and DNA and histone modifications. Several studies have uncovered that R-loops contribute to fundamental biological processes in various organisms. Paradoxically, although they do play essential positive functions required for important biological processes, they can also contribute to DNA damage and genome instability. Recent evidence suggests that R-loops are involved in a number of human diseases, including neurological disorders, cancer, and autoimmune diseases. This review focuses on the molecular basis for R-loop–mediated gene regulation and genomic instability and briefly discusses methods for identifying R-loops in vivo. It also highlights recent studies indicating the role of R-loops in DNA double-strand break repair with an updated view of much-needed future goals in R-loop biology.

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“…The physiological relevance of reduced SSA activity could affect other RAD52‐dependent events apart from the repair of DSBs by SSA per se. In addition to the repair of DSBs via SSA and HDR, hRAD52 has been implicated in break‐induced replication (BIR) (Sotiriou et al , 2016), transcription‐coupled homologous recombination (Teng et al , 2018), processing of stalled replication forks (Malacaria et al , 2019), mitotic DNA synthesis (MiDAS) (Bhowmick et al , 2016), and alternative lengthening of telomeres (Zhang et al , 2019), as well as several other pathways (Jalan et al , 2019). Thus, hRAD52 likely plays an important role in maintaining the viability of cancer cells under replication stress and could help explain the protective effect of hRAD52 S346X.…”

Section: Discussionmentioning

confidence: 99%

The RAD52 S346X variant reduces risk of developing breast cancer in carriers of pathogenic germline BRCA2 mutations

et al. 2020

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Women who carry pathogenic mutations in BRCA1 and BRCA2 have a lifetime risk of developing breast cancer of up to 80%. However, risk estimates vary in part due to genetic modifiers. We investigated the association of the RAD52 S346X variant as a modifier of the risk of developing breast and ovarian cancers in BRCA1 and BRCA2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2. The RAD52 S346X allele was associated with a reduced risk of developing breast cancer in BRCA2 carriers [per-allele hazard ratio (HR) = 0.69, 95% confidence interval (CI) 0.56-0.86; P = 0.0008] and to a lesser extent in BRCA1 carriers (per-allele HR = 0.78, 95% CI 0.64-0.97, P = 0.02). We examined how this variant affected DNA repair. Using a reporter system that measures repair of DNA double-strand breaks (DSBs) by single-strand annealing (SSA), expression of hRAD52 suppressed the loss of this repair in Rad52 À/À mouse embryonic stem cells. When hRAD52 S346X was expressed in these cells, there was a significantly reduced frequency of SSA. Interestingly, expression of hRAD52 S346X also reduced the stimulation of SSA observed upon depletion of BRCA2, demonstrating the reciprocal roles for RAD52 and BRCA2 in the control of DSB repair by SSA. From an immunofluorescence analysis, we observed little nuclear localization of the mutant protein as compared to the wild-type; it is likely that the reduced nuclear levels of RAD52 S346X explain the diminished DSB repair by SSA. Altogether, we identified a genetic modifier that protects against breast cancer in women who carry pathogenic mutations in BRCA2 (P = 0.0008) and to a lesser extent BRCA1 (P = 0.02). This RAD52 mutation causes a reduction in DSB repair by SSA, suggesting that defects in RAD52-dependent DSB repair are linked to reduced tumor risk in BRCA2-mutation carriers.Abbreviations CI, confidence interval; CIMBA, Consortium of Investigators of Modifiers of BRCA1/2; DSB, DNA double-strand break; GFP, green fluorescent protein; HDR, homology-directed repair; HR, hazard ratio; MAF, minor allele frequency; mESCs, mouse embryonic stem cells;

show abstract

ROS-induced R loops trigger a transcription-coupled but BRCA1/2-independent homologous recombination pathway through CSB

Cited by 134 publications

References 41 publications

Arginine methylation of the DDX 5 helicase RGG / RG motif by PRMT 5 regulates resolution of RNA:DNA hybrids

Arginine methylation of the DDX 5 helicase RGG / RG motif by PRMT 5 regulates resolution of RNA:DNA hybrids

The balancing act of R-loop biology: The good, the bad, and the ugly

The RAD52 S346X variant reduces risk of developing breast cancer in carriers of pathogenic germline BRCA2 mutations

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