Browse
Submit Data
Databases
API
Help

Dataset Information

26 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2.

ABSTRACT: Homologous recombination (HR) and non-homologous end joining (NHEJ) represent distinct pathways for repairing DNA double-strand breaks (DSBs). Previous work implicated Artemis and ATM in an NHEJ-dependent process, which repairs a defined subset of radiation-induced DSBs in G1-phase. Here, we show that in G2, as in G1, NHEJ represents the major DSB-repair pathway whereas HR is only essential for repair of approximately 15% of X- or gamma-ray-induced DSBs. In addition to requiring the known HR proteins, Brca2, Rad51 and Rad54, repair of radiation-induced DSBs by HR in G2 also involves Artemis and ATM suggesting that they promote NHEJ during G1 but HR during G2. The dependency for ATM for repair is relieved by depleting KAP-1, providing evidence that HR in G2 repairs heterochromatin-associated DSBs. Although not core HR proteins, ATM and Artemis are required for efficient formation of single-stranded DNA and Rad51 foci at radiation-induced DSBs in G2 with Artemis function requiring its endonuclease activity. We suggest that Artemis endonuclease removes lesions or secondary structures, which inhibit end resection and preclude the completion of HR or NHEJ.

SUBMITTER: Beucher A

PROVIDER: S-EPMC2752027 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Radiation-induced double-strand breaks require ATM but not Artemis for homologous recombination during S-phase.

Project description:Double-strand breaks (DSBs) are repaired by two distinct pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). The endonuclease Artemis and the PIK kinase Ataxia-Telangiectasia Mutated (ATM), mutated in prominent human radiosensitivity syndromes, are essential for repairing a subset of DSBs via NHEJ in G1 and HR in G2. Both proteins have been implicated in DNA end resection, a mandatory step preceding homology search and strand pairing in HR. Here, we show that during S-phase Artemis but not ATM is dispensable for HR of radiation-induced DSBs. In replicating AT cells, numerous Rad51 foci form gradually, indicating a Rad51 recruitment process that is independent of ATM-mediated end resection. Those DSBs decorated with Rad51 persisted through S- and G2-phase indicating incomplete HR resulting in unrepaired DSBs and a pronounced G2 arrest. We demonstrate that in AT cells loading of Rad51 depends on functional ATR/Chk1. The ATR-dependent checkpoint response is most likely activated when the replication fork encounters radiation-induced single-strand breaks leading to generation of long stretches of single-stranded DNA. Together, these results provide new insight into the role of ATM for initiation and completion of HR during S- and G2-phase. The DSB repair defect during S-phase significantly contributes to the radiosensitivity of AT cells.

| S-EPMC3458552 | biostudies-literature

ATM release at resected double-strand breaks provides heterochromatin reconstitution to facilitate homologous recombination.

Project description:Non-homologous end-joining (NHEJ) and homologous recombination (HR) represent the two main pathways for repairing DNA double-strand breaks (DSBs). During the G2 phase of the mammalian cell cycle, both processes can operate and chromatin structure is one important factor which determines DSB repair pathway choice. ATM facilitates the repair of heterochromatic DSBs by phosphorylating and inactivating the heterochromatin building factor KAP-1, leading to local chromatin relaxation. Here, we show that ATM accumulation and activity is strongly diminished at DSBs undergoing end-resection during HR. Such DSBs remain unrepaired in cells devoid of the HR factors BRCA2, XRCC3 or RAD51. Strikingly, depletion of KAP-1 or expression of phospho-mimic KAP-1 allows repair of resected DSBs in the absence of BRCA2, XRCC3 or RAD51 by an erroneous PARP-dependent alt-NHEJ process. We suggest that DSBs in heterochromatin elicit initial local heterochromatin relaxation which is reversed during HR due to the release of ATM from resection break ends. The restored heterochromatic structure facilitates HR and prevents usage of error-prone alternative processes.

| S-EPMC3731223 | biostudies-literature

Homologous recombination repairs secondary replication induced DNA double-strand breaks after ionizing radiation.

Project description:Ionizing radiation (IR) produces direct two-ended DNA double-strand breaks (DSBs) primarily repaired by non-homologous end joining (NHEJ). It is, however, well established that homologous recombination (HR) is induced and required for repair of a subset of DSBs formed following IR. Here, we find that HR induced by IR is drastically reduced when post-DNA damage replication is inhibited in mammalian cells. Both IR-induced RAD51 foci and HR events in the hprt gene are reduced in the presence of replication polymerase inhibitor aphidicolin (APH). Interestingly, we also detect reduced IR-induced toxicity in HR deficient cells when inhibiting post-DNA damage replication. When studying DSB formation following IR exposure, we find that apart from the direct DSBs the treatment also triggers formation of secondary DSBs peaking at 7-9 h after exposure. These secondary DSBs are restricted to newly replicated DNA and abolished by inhibiting post-DNA damage replication. Further, we find that IR-induced RAD51 foci are decreased by APH only in cells replicating at the time of IR exposure, suggesting distinct differences between IR-induced HR in S- and G2-phases of the cell cycle. Altogether, our data indicate that secondary replication-associated DSBs formed following exposure to IR are major substrates for IR-induced HR repair.

| S-EPMC3413124 | biostudies-literature

Homologous recombination repair intermediates promote efficient de novo telomere addition at DNA double-strand breaks.

Project description:The healing of broken chromosomes by de novo telomere addition, while a normal developmental process in some organisms, has the potential to cause extensive loss of heterozygosity, genetic disease, or cell death. However, it is unclear how de novo telomere addition (dnTA) is regulated at DNA double-strand breaks (DSBs). Here, using a non-essential minichromosome in fission yeast, we identify roles for the HR factors Rqh1 helicase, in concert with Rad55, in suppressing dnTA at or near a DSB. We find the frequency of dnTA in rqh1? rad55? cells is reduced following loss of Exo1, Swi5 or Rad51. Strikingly, in the absence of the distal homologous chromosome arm dnTA is further increased, with nearly half of the breaks being healed in rqh1? rad55? or rqh1? exo1? cells. These findings provide new insights into the genetic context of highly efficient dnTA within HR intermediates, and how such events are normally suppressed to maintain genome stability.

| S-EPMC7026635 | biostudies-literature

Homologous recombination and the repair of DNA double-strand breaks.

Project description:Homologous recombination enables the cell to access and copy intact DNA sequence information in trans, particularly to repair DNA damage affecting both strands of the double helix. Here, we discuss the DNA transactions and enzymatic activities required for this elegantly orchestrated process in the context of the repair of DNA double-strand breaks in somatic cells. This includes homology search, DNA strand invasion, repair DNA synthesis, and restoration of intact chromosomes. Aspects of DNA topology affecting individual steps are highlighted. Overall, recombination is a dynamic pathway with multiple metastable and reversible intermediates designed to achieve DNA repair with high fidelity.

| S-EPMC6036207 | biostudies-literature

Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks.

Project description:Although both homologous recombination (HR) and nonhomologous end joining can repair DNA double-strand breaks (DSBs), the mechanisms by which one of these pathways is chosen over the other remain unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation-sequencing (ChIP-seq) to analyze repair of multiple DSBs induced throughout the human genome, 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 are targeted to HR repair via the transcription elongation-associated mark trimethylated histone H3 K36. Concordantly, depletion of SETD2, the main H3 K36 trimethyltransferase, severely impedes HR at such DSBs. Our study thereby demonstrates a primary role in DSB repair of the chromatin context in which a break occurs.

| S-EPMC4300393 | biostudies-literature

CRISPR-induced double-strand breaks trigger recombination between homologous chromosome arms.

Project description:CRISPR-Cas9-based genome editing has transformed the life sciences, enabling virtually unlimited genetic manipulation of genomes: The RNA-guided Cas9 endonuclease cuts DNA at a specific target sequence and the resulting double-strand breaks are mended by one of the intrinsic cellular repair pathways. Imprecise double-strand repair will introduce random mutations such as indels or point mutations, whereas precise editing will restore or specifically edit the locus as mandated by an endogenous or exogenously provided template. Recent studies indicate that CRISPR-induced DNA cuts may also result in the exchange of genetic information between homologous chromosome arms. However, conclusive data of such recombination events in higher eukaryotes are lacking. Here, we show that in Drosophila, the detected Cas9-mediated editing events frequently resulted in germline-transmitted exchange of chromosome arms-often without indels. These findings demonstrate the feasibility of using the system for generating recombinants and also highlight an unforeseen risk of using CRISPR-Cas9 for therapeutic intervention.

| S-EPMC6587125 | biostudies-literature

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks.

Project description:DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability.

| S-EPMC2847229 | biostudies-literature

GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination.

Project description:RAD51 is a key factor in homologous recombination (HR) and plays an essential role in cellular proliferation by repairing DNA damage during replication. The assembly of RAD51 at DNA damage is strictly controlled by RAD51 mediators, including BRCA1 and BRCA2. We found that human RAD51 directly binds GEMIN2/SIP1, a protein involved in spliceosome biogenesis. Biochemical analyses indicated that GEMIN2 enhances the RAD51-DNA complex formation by inhibiting RAD51 dissociation from DNA, and thereby stimulates RAD51-mediated homologous pairing. GEMIN2 also enhanced the RAD51-mediated strand exchange, when RPA was pre-bound to ssDNA before the addition of RAD51. To analyze the function of GEMIN2, we depleted GEMIN2 in the chicken DT40 line and in human cells. The loss of GEMIN2 reduced HR efficiency and resulted in a significant decrease in the number of RAD51 subnuclear foci, as observed in cells deficient in BRCA1 and BRCA2. These observations and our biochemical analyses reveal that GEMIN2 regulates HR as a novel RAD51 mediator.

| S-EPMC2926616 | biostudies-literature

Recruitment of RBM6 to DNA Double-Strand Breaks Fosters Homologous Recombination Repair.

Project description:DNA double-strand breaks (DSBs) are highly toxic lesions that threaten genome integrity and cell survival. To avoid harmful repercussions of DSBs, a wide variety of DNA repair factors are recruited to execute DSB repair. Previously, we demonstrated that RBM6 splicing factor facilitates homologous recombination (HR) of DSB by regulating alternative splicing-coupled nonstop-decay of the HR protein APBB1/Fe65. Here, we describe a splicing-independent function of RBM6 in promoting HR repair of DSBs. We show that RBM6 is recruited to DSB sites and PARP1 activity indirectly regulates RBM6 recruitment to DNA breakage sites. Deletion mapping analysis revealed a region containing five glycine residues within the G-patch domain that regulates RBM6 accumulation at DNA damage sites. We further ascertain that RBM6 interacts with Rad51, and this interaction is attenuated in RBM6 mutant lacking the G-patch domain (RBM6del(G-patch)). Consequently, RBM6del(G-patch) cells exhibit reduced levels of Rad51 foci after ionizing radiation. In addition, while RBM6 deletion mutant lacking the G-patch domain has no detectable effect on the expression levels of its splicing targets Fe65 and Eya2, it fails to restore the integrity of HR. Altogether, our results suggest that RBM6 recruitment to DSB promotes HR repair, irrespective of its splicing activity.HIGHLIGHTSPARP1 activity indirectly regulates RBM6 recruitment to DNA damage sites.Five glycine residues within the G-patch domain of RBM6 are critical for its recruitment to DNA damage sites, but dispensable for its splicing activity.RBM6 G-patch domain fosters its interaction with Rad51 and promotes Rad51 foci formation following irradiation.RBM6 recruitment to DSB sites underpins HR repair.

| S-EPMC10038030 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data