BRCA2 prevents R-loop accumulation and associates with TREX-2 mRNA export factor PCID2

Bhatia, Vaibhav; Barroso, Sónia; García-Rubio, María L.; Tumini, Emanuela; Herrera-Moyano, Emilia; Aguilera, Andrés

doi:10.1038/nature13374

Cited by 459 publications

(545 citation statements)

References 33 publications

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“…2B and 3B). Moreover, other familial ALS proteins are involved in the prevention/repair of this type of damage (16,24), and BRCA1, which colocalizes with FUS and likely TDP43 at post-UV RNA Pol II DNA damage foci, also participates in the prevention and/or repair of R loop-associated DNA damage (18,26,27). Thus, we asked whether FUS and/or TDP43 participate in the prevention or repair of R loop-associated DNA damage by searching for a decrease in DNA damage following FUS or TDP43 depletion in cells that express ectopic RNASEH1.…”

Section: Depletion Of Fus and Tdp43 Leads To Excessive Transcription mentioning

confidence: 99%

Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage

Hill

Mordes

Cameron

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

107

119

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Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron dysfunction disease that leads to paralysis and death. There is currently no established molecular pathogenesis pathway. Multiple proteins involved in RNA processing are linked to ALS, including FUS and TDP43, and we propose a disease mechanism in which loss of function of at least one of these proteins leads to an accumulation of transcription-associated DNA damage contributing to motor neuron cell death and progressive neurological symptoms. In support of this hypothesis, we find that FUS or TDP43 depletion leads to increased sensitivity to a transcription-arresting agent due to increased DNA damage. Thus, these proteins normally contribute to the prevention or repair of transcription-associated DNA damage. In addition, both FUS and TDP43 colocalize with active RNA polymerase II at sites of DNA damage along with the DNA damage repair protein, BRCA1, and FUS and TDP43 participate in the prevention or repair of R loopassociated DNA damage, a manifestation of aberrant transcription and/or RNA processing. Gaining a better understanding of the role(s) that FUS and TDP43 play in transcription-associated DNA damage could shed light on the mechanisms underlying ALS pathogenesis.A myotrophic lateral sclerosis (ALS) is a disease of both upper and lower motor neuron dysfunction that leads to progressive paralysis and eventually death due to respiratory failure. No single model of ALS disease pathogenesis has been revealed; however, multiple disease-associated genes are known (1). The variation in function of these genes suggests that there may be multiple ALS molecular subtypes. That said, the familial ALS gene product list is also enriched in protein groups with related functions.Mutations in multiple RNA processing genes, including FUS (FUS RNA-binding protein), TDP43 (TAR DNA-binding protein), SETX (senataxin), TAF15 (TATA box-binding protein-associated factor 15), EWSR1 (EWS RNA-binding protein 1), HNRNPA1 (heterogeneous nuclear ribonucleoprotein A1), and HNRNPA2B1 (heterogeneous nuclear ribonucleoprotein A2/B1), among others, give rise to familial ALS (fALS) (1). FUS and TDP43 are currently the best studied, given that mutations in these genes lead to classic histologic findings in ALS neural tissue and that similar histologic findings can be found in neural tissue of sporadic ALS cases (2). At autopsy, cytoplasmic TDP43 inclusions are found in ALS motor neurons from patients with (i) TDP43 mutations and (ii) sporadic ALS (i.e., patients with no FUS, TDP43, or other known pathogenic mutation) (2). They are also present in neurons in various parts of the brains of patients with either sporadic or familial versions of the ALS-related disorder, fronto-temporal lobar dementia (FTLD) (2). At autopsy, FUS inclusions were detected in the cytoplasm of motor neurons of FUS mutation-bearing fALS patients and in the cytoplasm of neurons in various regions of the brain in some FTLD patients (2).FUS and TDP43 exhibit a wide range of functions, most of which involv...

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Section: Depletion Of Fus and Tdp43 Leads To Excessive Transcription mentioning

confidence: 99%

Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage

Hill

Mordes

Cameron

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

107

119

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“…In vitro, converging replisomes continue through their meeting point as one replisome displaces the other, resulting in over-replication, or a third copy, of the region where the forks meet (8). Complicating the process of genomic doubling even further, several studies have suggested that illegitimate initiations of replication frequently occur at single-strand nicks, gaps, Dloops, and R-loops throughout the genomes of both prokaryotes and eukaryotes (9)(10)(11)(12)(13)(14). Similar to when replication forks continue through a previously replicated template, each of these events would generate a third copy of the chromosomal region where the event occurs.…”

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confidence: 99%

Completion of DNA replication in Escherichia coli

Wendel

Courcelle

2014

Proc. Natl. Acad. Sci. U.S.A.

134

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The mechanism by which cells recognize and complete replicated regions at their precise doubling point must be remarkably efficient, occurring thousands of times per cell division along the chromosomes of humans. However, this process remains poorly understood. Here we show that, in Escherichia coli, the completion of replication involves an enzymatic system that effectively counts pairs and limits cellular replication to its doubling point by allowing converging replication forks to transiently continue through the doubling point before the excess, over-replicated regions are incised, resected, and joined. Completion requires RecBCD and involves several proteins associated with repairing double-strand breaks including, ExoI, SbcDC, and RecG. However, unlike double-strand break repair, completion occurs independently of homologous recombination and RecA. In some bacterial viruses, the completion mechanism is specifically targeted for inactivation to allow over-replication to occur during lytic replication. The results suggest that a primary cause of genomic instabilities in many double-strand-break-repair mutants arises from an impaired ability to complete replication, independent from DNA damage.replication completion | double-strand break repair | RecBCD | homologous recombination | SbcDC

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“…Two regions upstream of the I-SceI cut site, P2 and P3, were examined. For each region, the relative enrichment of DNA-RNA hybrids was normalized to the signals obtained from input genomic DNA as well as the SNRPN negative-control region, as previously described for DRIP analysis (27)(28)(29). The average percent IPs in control (scrambled siRNA-transfected) cells for the SNRPN (negative control), P2, and P3 loci were 0.06%, 0.55%, and 2.42%, respectively.…”

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confidence: 99%

“…Twenty-four hours after DNA transfection, 4-hydroxytamoxifen (Sigma) was added to a final concentration of 5 M. Cells were incubated for 4 h and then harvested. The procedure for preparing nucleic acids for DNA-RNA immunoprecipitation (DRIP) analysis has been reported previously (27). Briefly, nucleic acids (genomic DNA along with RNA) were purified by lysing cell pellets in 1% SDS and digesting the lysates overnight with 300 g/ml proteinase K. Nucleic acids were extracted using a neutral-pH phenol-chloroform (1:1) mixture followed by ethanol precipitation.…”

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confidence: 99%

DEAD Box 1 Facilitates Removal of RNA and Homologous Recombination at DNA Double-Strand Breaks

Germain

Poon

et al. 2016

Molecular and Cellular Biology

132

138

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Although RNA and RNA-binding proteins have been linked to double-strand breaks (DSBs), little is known regarding their roles in the cellular response to DSBs and, if any, in the repair process. Here, we provide direct evidence for the presence of RNA-DNA hybrids at DSBs and suggest that binding of RNA to DNA at DSBs may impact repair efficiency. Our data indicate that the RNAunwinding protein DEAD box 1 (DDX1) is required for efficient DSB repair and cell survival after ionizing radiation (IR), with depletion of DDX1 resulting in reduced DSB repair by homologous recombination (HR). While DDX1 is not essential for end resection, a key step in homology-directed DSB repair, DDX1 is required for maintenance of the single-stranded DNA once generated by end resection. We show that transcription deregulation has a significant effect on DSB repair by HR in DDX1-depleted cells and that RNA-DNA duplexes are elevated at DSBs in DDX1-depleted cells. Based on our combined data, we propose a role for DDX1 in resolving RNA-DNA structures that accumulate at DSBs located at sites of active transcription. Our findings point to a previously uncharacterized requirement for clearing RNA at DSBs for efficient repair by HR. DEAD box proteins are a family of putative RNA helicases that function by altering RNA secondary structure. This protein family has been implicated in all aspects of RNA metabolism. The DEAD box 1 gene (DDX1) is a widely expressed gene that is misexpressed in a number of cancers, including retinoblastoma, neuroblastoma, and breast cancer (1, 2). Knockout of DDX1 leads to early embryonic lethality in mice and severely reduced fertility in flies (3, 4). DDX1 is involved in the transport of RNAs from the nucleus to the cytoplasm and regulates cytoplasmic localization of the splicing-regulatory protein KSRP (5). In neurons, DDX1 resides in RNA-transporting granules, cytoplasmic organelles that regulate the localization and expression of target mRNAs (6, 7). DDX1 has also been identified as a core subunit of the human tRNA ligase complex which is essential for tRNA splicing (8).In addition to its roles in RNA metabolism, DDX1 has been implicated in the cellular response to DNA double-strand breaks (DSBs). Upon treatment of cells with ionizing radiation (IR), DDX1 rapidly accumulates at a subset of DNA DSBs (ϳ30%), where it forms IR-induced foci that colocalize with ␥-H2AX, a marker for DSBs (9). DDX1 coimmunoprecipitates with the MRN (MRE11-RAD50-NBS1) complex, the early sensor of DNA DSBs, and ATM (ataxia telangiectasia mutated) protein, the key transducer of the signaling cascade in response to DSBs (10, 11). DSBs induce DDX1 phosphorylation in an ATM-dependent manner. Notably, IR-induced DDX1 foci are lost when cells are treated with RNase H, an enzyme that specifically digests RNA from RNA-DNA hybrids (9). These results suggest that RNA-DNA double-stranded structures are required for the presence and/or retention of DDX1 at DSBs. In line with this observation, biochemical analysis has shown that DDX1 can unwind both...

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BRCA2 prevents R-loop accumulation and associates with TREX-2 mRNA export factor PCID2

Cited by 459 publications

References 33 publications

Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage

Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage

Completion of DNA replication in Escherichia coli

DEAD Box 1 Facilitates Removal of RNA and Homologous Recombination at DNA Double-Strand Breaks

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