Biology subjects

Coulombe, Y.

Publications and source records attributed to Coulombe, Y..

4 recordsLinked to original sources

bioRxiv2025

The Ski2 helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses

The activating signal co-integrator 1 complex subunit 3 (ASCC3), the largest subunit of ASCC, is one of two Ski2-like helicases with duplicated helicase cassettes encoded by the human genome. ASCC3 has been implicated in transcriptional regulation, alkylation damage repair, and ribosome quality control. In addition, published proteomics studies suggest that ASCC3 is associated with stalled forks. However, little is known about its role in replication stress. Here we report that ASCC3 is recruited to stalled forks by its binding partner ASCC2, whose recruitment to stalled forks is dependent upon both its ubiquitin binding activity and polyubiquitylation of PCNA at K164 that is catalyzed by SHPRH, HLTF, and RFWD3. In response to replication stress. ASCC3 unwinds DNA, generating both parental and nascent ssDNA. This unwinding activity is required to promote fork reversal. We demonstrate that ASCC3 also interacts with RPA and stimulates RPA accumulation on ssDNA upon replication stress, promoting ATR activation. Furthermore, ASCC3 functions to antagonize RAD51-mediated recombination and prevents accumulation of chromosome breaks/gaps and mis-segregation in response to replication stress. Our work underscores a previously uncharacterized but critical role of ASCC3 in controlling multiple replication stress responses to maintain genomic stability.

molecular biology↗

bioRxiv2018

MRE11-RAD50-NBS1 activates Fanconi Anemia R-loop suppression at transcription-replication conflicts

Ectopic R-loop accumulation causes DNA replication stress and genome instability. To avoid these outcomes, cells possess a range of anti-R-loop mechanisms, including RNaseH that degrades the RNA moiety in R-loops. To comprehensively identify anti-R-loop mechanisms, we performed a genome-wide trigenic interaction screen in yeast lacking RNH1 and RNH201. We identified >100 genes critical for fitness in the absence of RNaseH, which were enriched for DNA replication fork maintenance factors such as RAD50. We show in yeast and human cells that R-loops accumulate during RAD50 depletion. In human cancer cell models, we find that RAD50 and its partners in the MRE11-RAD50-NBS1 complex regulate R-loop-associated DNA damage and replication stress. We show that a non-nucleolytic function of MRE11 is important for R-loop suppression via activation of PCNA-ubiquitination by RAD18 and recruiting anti-R-loop helicases in the Fanconi Anemia pathway. This work establishes a novel role for MRE11-RAD50-NBS1 in directing tolerance mechanisms of transcription-replication conflicts.

cell biology↗

bioRxiv2018

Arginine methylation of DDX5 RGG/RG motif by PRMT5 regulates RNA/DNA resolution.

Aberrant transcription-associated RNA:DNA hybrid (R-loop) formation often lead to catastrophic conflicts during replication resulting in DNA double strand breaks and genome instability. To prevent such conflicts, these hybrids require dissolution by helicases and/or RNaseH. Little information is known about how these helicases are regulated. Herein, we identify DDX5, an RGG/RG motif containing DEAD-box family of RNA helicase, as a crucial player in R-loop resolution. We define at the mechanistic level the function of DDX5 in R-loop resolution. In vitro, recombinant DDX5 resolves R-loops in an ATP-dependent manner leading to R-loop degradation by the XRN2 exoribonuclease. DDX5 deficient cells accumulated R-loops at loci known to form R-loops using RNA:DNA immunoprecipitation (DRIP)-qPCR and increased RNaseH sensitive RAD51 foci. PRMT5, an arginine methyltransferase, associated with DDX5 and methylated its RGG/RG motif. This motif was required to associate with XRN2 and resolve cellular R-loops. Furthermore, PRMT5 deficient cells accumulated R-loops, as detected by DRIP-qPCR resulting in increased gH2AX foci. Our findings define a new mechanism by which an RNA helicase, DDX5, is modulated by arginine methylation to resolve R-loops.

molecular biology↗

bioRxiv2017

Conserved roles of RECQ-like helicases Sgs1 and BLM in preventing R-loop induced genome instability

Sgs1 is a yeast DNA helicase functioning in DNA replication and repair, and is the orthologue of the human Blooms syndrome helicase BLM. Here we analyze the mutation signature associated with SGS1 deletion in yeast, and find frequent copy number changes flanked by regions of repetitive sequence and high R-loop forming potential. We show that loss of SGS1 increases R-loop accumulation and sensitizes cells to replication-transcription collisions. Accordingly, in sgs1{Delta} cells the genome-wide distribution of R-loops shifts to known sites of Sgs1 action, replication pausing regions, and to long genes. Depletion of the orthologous BLM helicase from human cancer cells also increases R-loop levels, and R-loop-associated genome instability. In support of a direct effect, BLM is found physically proximal to DNA:RNA hybrids in human cells, and can efficiently unwind R-loops in vitro. Together our data describe a conserved role for Sgs1/BLM in R-loop suppression and support an increasingly broad view of DNA repair and replication fork stabilizing proteins as modulators of R-loop mediated genome instability.

cell biology↗