RAD51 Interconnects Between DNA Replication, DNA Repair and Immunity
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE95050 | GEO | 2017/02/18
SECONDARY ACCESSION(S): PRJNA375818
REPOSITORIES: GEO
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Project description:The proper maintenance of genetic material is essential for the survival of living organisms. One of the main safeguards of genome stability is homologous recombination involved in the faithful repair of DNA double-strand breaks, the restoration of collapsed replication forks, and the bypass of replication barriers. Homologous recombination relies on the formation of Rad51 nucleofilaments which are responsible for the homology-based interactions between DNA strands. Here we demonstrate that without the regulation of these filaments by Srs2 and Rad54, which are known to remove Rad51 from single-stranded and double-stranded DNA respectively, the filaments strongly inhibit damage-associated DNA synthesis during DNA repair. Furthermore, this regulation is essential for cell survival under normal growth conditions as in the srs2Δ rad54Δ mutants unregulated Rad51 nucleofilaments cause activation of the DNA damage checkpoint, formation of mitotic bridges and loss of genetic material. These genome instability features may stem from the problems at stalled replication forks as the lack of Srs2 and Rad54 in the presence of Rad51 nucleofilaments impedes cell recovery from replication stress. This study demonstrates that the timely and efficient disassembly of recombination machinery is essential for genome maintenance and cell survival.
2023-03-16 | GSE227383 | GEO
Project description:Cells have developed effective mechanisms, namely homologous recombination (HR) and non-homologous end-joining (NHEJ), to repair DNA double-strand breaks (DSBs), which are considered to be the most deleterious type of damage that can challenge genome integrity. While these pathways coexist to repair DSBs, the mechanisms by which one of these pathways is chosen to repair a particular DSB remain unclear. Here, we show that the chromatin context in which a break occurs participates in this choice and that transcriptionnaly active chromatin channels repair to HR. By using a human cell line expressing a restriction enzyme fused to the ligand binding domain of the oestrogen receptor (AsiSI-ER)2,3, together with a genome wide chromatin immunoprecipitation-sequencing (ChIP-seq) approach, we establish that distinct DSBs induced across the genome are not necessarily repaired by the same pathway. Indeed, we identify an HR-prone subset of DSBs that recruit the HR protein RAD51, undergo resection, and rely on RAD51 for efficient repair. These DSBs are located in actively transcribed genes, and repair at such DSBs can be switched to RAD51-independent repair pathway upon transcriptional inhibition. Moreover, we show that HR is targeted to transcribed loci thanks to the elongation-associated H3K36me3 histone mark. Indeed depletion of HYPB, the main H3K36 tri methyltransferase severally impedes the use of HR at those DSBs. Our study, thereby demonstrates a clear role for chromatin in DSB repair pathway choice in human cells.
2014-03-23 | E-MTAB-1241 | biostudies-arrayexpress
Project description:Homologous recombination (HR) plays an essential role in the maintenance of genome stability by promoting the repair of cytotoxic DNA double strand breaks (DSBs). More recently, the HR pathway has emerged as an integral component of the response to replication stress, in part by protecting stalled replication forks from nucleolytic degradation. In that regard, the mammalian RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) have been involved in both HR-mediated DNA repair and collapsed replication fork resolution. Still, it remains largely obscure how they participate in both processes, thereby maintaining genome stability and preventing cancer development. To gain better insight into their contribution in cellulo, we mapped the proximal interactome of the classical RAD51 paralogs using the BioID approach. Aside from identifying the well-established BCDX2 and CX3 sub-complexes, the spliceosome machinery emerged as an integral component of our proximal mapping, suggesting a crosstalk between this pathway and the RAD51 paralogs. Furthermore, we noticed that factors involved RNA metabolic pathways are significantly modulated within the BioID of the classical RAD51 paralogs upon exposure to hydroxyurea (HU), pointing towards a direct contribution of RNA processing during replication stress. Importantly, several members of these pathways have prognosis potential in breast cancer (BC), where their RNA expression correlates with poorer patient outcome. Collectively, this study uncovers novel functionally relevant partners of the different RAD51 paralogs in the maintenance of genome stability and could be used as biomarker for the prognosis of BC.
2022-05-20 | MSV000089507 | MassIVE
Project description:Early relapse after platinum chemotherapy in epithelial ovarian cancer (EOC) portends poor survival. A-priori identification of platinum-resistance is therefore crucial to improve on standard first-line carboplatin-paclitaxel treatment. The DNA repair pathway Homologous Recombination (HR) repairs platinum-induced damage, and the HR recombinase RAD51 is overexpressed in cancer. We therefore designed a REMARK-compliant study of pre-treatment RAD51 expression in EOC, using fluorescent quantitative immunohistochemistry (qIHC) to overcome challenges in quantitation of protein-expression in-situ. In a discovery cohort (n=284), RAD51-High tumours had shorter progression-free and overall survival compared to RAD51-Low cases in univariate and multivariate analyses. The association of RAD51 with relapse/survival was validated in a carboplatin-monotherapy SCOTROC4 clinical trial cohort (n=264), and was predominantly noted in HR-proficient cancers (Myriad HRDscore<42). Interestingly, overexpression of RAD51 modified expression of immune-regulatory pathways in-vitro, while high-RAD51 tumours showed exclusion of cytotoxic-T-cells in-situ. Our findings highlight RAD51 expression as a determinant of platinum resistance, and suggest possible roles for therapy to overcome immune exclusion in high-RAD51 EOC. The qIHC approach is generalizable to other proteins with a continuum instead of discrete/bimodal expression.
2021-02-11 | GSE166539 | GEO
Project description:Meiotic recombination is initiated by programmed DNA double-strand breaks (DSBs) and proceeds via binding of RPA, RAD51 and DMC1 to single-stranded DNA (ssDNA) substrates created after the formation of DSBs. Here, we report high-resolution in vivo maps of RPA and RAD51 binding in meiosis, mapping their binding locations and lifespans in a B6 and a genetically modified B6xCAST F1 mouse. We ascribe signals separately to the individual homologous chromosomes in the hybrid mouse, thereby separating the signal of binding to the chromosome where DSBs occurred and the chromosome that was used as template for repair. Together with super-resolution microscopy and DMC1 binding maps, we show that DMC1 and RAD51 have distinct spatial localization on ssDNA: whereas DMC1 binds near the break-site, RAD51 binds away from it. We characterize the D-loop, a critical intermediate bound by RPA, in vivo. These data show that DMC1, not RAD51, performs strand exchange in mammalian meiosis. We find that the localisation of D-loop intermediates is similar in crossover and non-crossover pathways, with a longer lifespan for crossover-destined intermediates. These findings answer long-standing questions about the molecular intermediates of recombination.
2020-06-30 | GSE143582 | GEO
Project description:The formation of RAD51/DMC1 filaments on single-stranded (ss)DNAs, which is essential for homology search and strand exchange in DNA double-strand break (DSB) repair and for the protection of stalled DNA replication forks, is tightly regulated in time and space. FIGNL1 AAA+++ ATPase plays positive and negative roles in the RAD51-mediated recombination in human cells. However, the role of FIGNL1 in gametogenesis remains unsolved. Here, we characterized a germ-line-specific conditional knockout (cKO) mouse of FIGNL1. The Fignl1 cKO male mice showed defective chromosome synapsis and impaired meiotic DSB repair with the accumulation of RAD51/DMC1 on chromosomes in mid-meiotic prophase I, supporting a role of FIGNL1 in a post-assembly stage of RAD51/DMC1 filaments. Fignl1 cKO spermatocytes accumulate RAD51 and DMC1 ensembles on chromosomes not only in early meiotic prophase I but also in meiotic S-phase. These RAD51/DMC1 assemblies are independent of meiotic DSB formation. Finally, we showed that purified FIGNL1 dismantles RAD51 filament on double-stranded (ds)DNA as well as ssDNA. These results suggest a critical role of FIGNL1 to limit the uncontrolled assembly of RAD51 and DMC1 on native dsDNAs during the meiotic S-phase and meiotic prophase I.
2023-03-31 | GSE227944 | GEO
Project description:DNA replication stress is an established driver of cancer-associated chromosomal rearrangements. Replication stress perturbs the duplication of late-replicating loci and activates a mitotic DNA repair pathway (termed MiDAS) for completion of replication. We here investigated RAD51-independent MiDAS.
2022-06-27 | GSE196278 | GEO
Project description:Mammalian meiotic recombination proceeds via repair of hundreds of programmed DNA double-strand breaks (DSBs). This process requires choreographed binding of RPA, DMC1 and RAD51 to single-stranded DNA (ssDNA) substrates and in vivo binding maps of these proteins provide insights into the underlying molecular mechanisms. When assayed in F1-hybrid mice, these maps can distinguish the broken chromosome from the homologous chromosome used as template for repair, which reveals further mechanistic detail and enables the structure of the recombination intermediates to be inferred. By applying CRISPR/Cas9 mutagenesis directly on F1-hybrid embryos, we have extended this powerful analysis technique to explore the molecular detail of recombination when a key component is knocked-out. As a proof-of-concept, we have generated biallelic knockouts of Dmc1 and built maps of meiotic binding of RAD51 and RPA in these knockout hybrid mice. Dmc1 mutants undergo meiotic arrest and comparison of these maps with those from wild-type mice is informative about the structure and timing of recombination intermediates in both genotypes. We confirm a complete abrogation of strand exchange in Dmc1 mutants, and observe a redistribution of RAD51 binding across both the distal and proximal ends of the resected DNA. We observe unexpected RPA and DMC1 binding in the wild-type, which suggests multiple rounds of strand invasion with template-switching in mouse. The methodology used involves direct phenotyping of hybrid “founder” mice following CRISPR mutagenesis and provides a high-throughput approach for the analysis of gene function during meiotic recombination, at low animal cost.
2023-10-16 | GSE239997 | GEO
Project description:RAD51 protein is an evolutionarily conserved recombinase that plays a central role in homologous recombination (HR) and DNA double strand break (DSB) repair. RAD51 inactivation by small molecules has been proposed as a strategy to impair the BRCA2/RAD51 binding and, ultimately, the HR pathway, with the aim to make cancer cells more sensitive to PARP inhibitors (PARPi). This strategy, which mimics a synthetic lethality (SL) approach, has been successfully assayed in vitro by using myr-BRC4, a peptide derived from the fourth BRC repeat of BRCA2, being the strongest reported natural RAD51 binder. The present study applies a method to obtain a proteomic fingerprint for RAD51 inhibition by the myr-BRC4 peptide (designed for a more efficient cell entry) using a mass spectroscopy (MS) proteomic approach. We performed a comparative proteomic profiling of the myr-BRC4 treated vs. untreated BxPC-3 pancreatic cancer cells and evaluated the differential expression of proteins. Among the identified proteomic hits, we focused our attention on proteins shared by both the RAD51 and the BRCA2 interactomes, and on those whose reduction showed high statistical significance. Three downregulated proteins were identified (FANCI, FANCD2, and RPA3) and protein downregulation was confirmed through immunoblotting analysis, validating the MS approach. Our results suggest that, being a direct consequence of RAD51 inhibition, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring HR impairment.
2023-08-15 | PXD042696 | Pride
Project description:Joint DNA molecules are natural by-products of DNA replication and repair. Persistent joint molecules give rise to ultrafine DNA bridges (UFBs) in mitosis, which compromise sister chromatid separation. The DNA translocase PICH (ERCC6L) plays a central role in UFB resolution. To better understand the genetic context rendering cells dependent on PICH, a genome-wide loss-of-function screen was performed to identify the genetic contexts in which cells become dependent on PICH. In addition to genes involved in DNA cohesion, centromere stability and DNA damage repair, we identified the uncharacterized protein C1orf112. We find that C1orf112 interacts with and stabilizes the AAA+ ATPase FIGNL1. Inactivation of either C1orf112 or FIGNL1 resulted in UFB formation, prolonged retention of RAD51 on chromatin, impaired replication fork dynamics, and consequently impaired genome maintenance. Combined, our data reveal that inactivation of C1orf112 or FIGNL1 dysregulates RAD51 dynamics at replication forks, resulting in DNA replication defects, and a dependency on PICH to preserve cell viability.
2024-08-08 | PXD035876 | Pride