DNA Double Strand Break Repair Pathways in Response to Different Types of Ionizing Radiation
Ontology highlight
ABSTRACT:
SUBMITTER: van de Kamp G
PROVIDER: S-EPMC8514742 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Radiation-induced foci (RIF) are nuclear puncta visualized by immunostaining of proteins that regulate DNA double-strand break (DSB) repair after exposure to ionizing radiation. RIF are a standard metric for measuring DSB formation and repair in clinical, environmental and space radiobiology. The time course and dose dependence of their formation has great potential to predict in vivo responses to ionizing radiation, predisposition to cancer and probability of adverse reactions to radiotherapy. However, increasing complexity of experimentally and therapeutically setups (charged particle, FLASH …) is associated with several confounding factors that must be taken into account when interpreting RIF values. In this review, we discuss the spatiotemporal characteristics of RIF development after irradiation, addressing the common confounding factors, including cell proliferation and foci merging. We also describe the relevant endpoints and mathematical models that enable accurate biological interpretation of RIF formation and resolution. Finally, we discuss the use of RIF as a biomarker for quantification and prediction of in vivo radiation responses, including important caveats relating to the choice of the biological endpoint and the detection method. This review intends to help scientific community design radiobiology experiments using RIF as a key metric and to provide suggestions for their biological interpretation.
| S-EPMC8693576 | biostudies-literature
Project description:The choice of DNA double strand break (DSB) repair pathway is determined at the stage of DSB end resection. Resection was proposed to control the balance between the two major DSB repair pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). Here, we examined the regulation of DSB repair pathway choice at two-ended DSBs following ionizing radiation (IR) in G2 phase of the cell cycle. We found that cells pre-exposed to low-dose IR preferred to undergo HR following challenge IR in G2, whereas NHEJ repair kinetics in G1 were not affected by pre-IR treatment. Consistent with the increase in HR usage, the challenge IR induced Replication protein A (RPA) foci formation and RPA phosphorylation, a marker of resection, were enhanced by pre-IR. However, neither major DNA damage signals nor the status of core NHEJ proteins, which influence the choice of repair pathway, was significantly altered in pre-IR treated cells. Moreover, the increase in usage of HR due to pre-IR exposure was prevented by treatment with ATM inhibitor during the incubation period between pre-IR and challenge IR. Taken together, the results of our study suggest that the ATM-dependent damage response after pre-IR changes the cellular environment, possibly by regulating gene expression or post-transcriptional modifications in a manner that promotes resection.
| S-EPMC4380452 | biostudies-literature
Project description:Nicotinamide adenine dinucleotide (NAD) serves as a substrate for protein deacetylases sirtuins and poly(ADP-ribose) polymerases, which are involved in the regulation of DNA double-strand break (DSB) repair molecular machinery by various mechanisms. However, the impact of NAD bioavailability on DSB repair remains poorly characterized. Herein, using immunocytochemical analysis of γH2AX, a marker for DSB, we investigated the effect of the pharmacological modulation of NAD levels on DSB repair capacity in human dermal fibroblasts exposed to moderate doses of ionizing radiation (IR). We demonstrated that NAD boosting with nicotinamide riboside did not affect the efficiency of DSB elimination after the exposure of cells to IR at 1 Gy. Moreover, even after irradiation at 5 Gy, we did not observe any decrease in intracellular NAD content. We also showed that, when the NAD pool was almost completely depleted by inhibition of its biosynthesis from nicotinamide, cells were still able to eliminate IR-induced DSB, though the activation of ATM kinase, its colocalization with γH2AX and DSB repair capacity were reduced in comparison to cells with normal NAD levels. Our results suggest that NAD-dependent processes, such as protein deacetylation and ADP-ribosylation, are important but not indispensable for DSB repair induced by moderate doses of IR.
| S-EPMC10252650 | biostudies-literature
Project description:Bacterial pathogens rely on their DNA repair pathways to resist genomic damage inflicted by the host. DNA double-strand breaks (DSBs) are especially threatening to bacterial viability. DSB repair by homologous recombination (HR) requires nucleases that resect DSB ends and a strand exchange protein that facilitates homology search. RecBCD and RecA perform these functions in Escherichia coli and constitute the major pathway of error-free DSB repair. Mycobacteria, including the human pathogen M. tuberculosis, elaborate an additional error-prone pathway of DSB repair via non-homologous end-joining (NHEJ) catalysed by Ku and DNA ligase D (LigD). Little is known about the relative contributions of HR and NHEJ to mycobacterial chromosome repair, the factors that dictate pathway choice, or the existence of additional DSB repair pathways. Here we demonstrate that Mycobacterium smegmatis has three DSB repair pathway options: HR, NHEJ and a novel mechanism of single-strand annealing (SSA). Inactivation of NHEJ or SSA is compensated by elevated HR. We find that mycobacterial RecBCD does not participate in HR or confer resistance to ionizing radiation (IR), but is required for the RecA-independent SSA pathway. In contrast, the mycobacterial helicase-nuclease AdnAB participates in the RecA-dependent HR pathway, and is a major determinant of resistance to IR and oxidative DNA damage. These findings reveal distinctive features of mycobacterial DSB repair, most notably the dedication of the RecBCD and AdnAB helicase-nuclease machines to distinct repair pathways.
| S-EPMC3812669 | biostudies-literature
Project description:Vertebrate cells have evolved two major pathways for repairing DNA double-strand breaks (DSBs), homologous recombination (HR) and nonhomologous DNA end-joining (NHEJ). To investigate the role of DNA ligase IV (Lig4) in DSB repair, we knocked out the Lig4 gene (LIG4) in the DT40 chicken B-lymphocyte cell line. The LIG4(-/-) cells showed a marked sensitivity to X-rays, bleomycin, and VP-16 and were more x-ray-sensitive in G(1) than late S or G(2)/M, suggesting a critical role of Lig4 in DSB repair by NHEJ. In support of this notion, HR was not impaired in LIG4(-/-) cells. LIG4(-/-) cells were more x-ray-sensitive when compared with KU70(-/-) DT40 cells, particularly at high doses. Strikingly, however, the x-ray sensitivity of KU70(-/-)/LIG4(-/-) double-mutant cells was essentially the same as that of KU70(-/-) cells, showing that Lig4 deficiency has no effect in the absence of Ku. These results indicate that Lig4 is exclusively required for the Ku-dependent NHEJ pathway of DSB repair and that other DNA ligases (I and III) do not substitute for this function. Our data may explain the observed severe phenotype of Lig4-deficient mice as compared with Ku-deficient mice.
| S-EPMC59776 | biostudies-literature
Project description:DNA double-strand breaks (DSBs) are the most dangerous type of DNA damage because they can result in the loss of large chromosomal regions. In all mammalian cells, DSBs that occur throughout the cell cycle are repaired predominantly by the non-homologous DNA end joining (NHEJ) pathway. Defects in NHEJ result in sensitivity to ionizing radiation and the ablation of lymphocytes. The NHEJ pathway utilizes proteins that recognize, resect, polymerize and ligate the DNA ends in a flexible manner. This flexibility permits NHEJ to function on a wide range of DNA-end configurations, with the resulting repaired DNA junctions often containing mutations. In this Review, we discuss the most recent findings regarding the relative involvement of the different NHEJ proteins in the repair of various DNA-end configurations. We also discuss the shunting of DNA-end repair to the auxiliary pathways of alternative end joining (a-EJ) or single-strand annealing (SSA) and the relevance of these different pathways to human disease.
| S-EPMC7062608 | biostudies-literature
Project description:Manganese is an essential trace element; nevertheless, on conditions of overload, it becomes toxic, with neurotoxicity being the main concern. Chromate is a well-known human carcinogen. The underlying mechanisms seem to be oxidative stress as well as direct DNA damage in the case of chromate, but also interactions with DNA repair systems in both cases. However, the impact of manganese and chromate on DNA double-strand break (DSB) repair pathways is largely unknown. In the present study, we examined the induction of DSB as well as the effect on specific DNA DSB repair mechanisms, namely homologous recombination (HR), non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ). We applied DSB repair pathway-specific reporter cell lines, pulsed field gel electrophoresis as well as gene expression analysis, and investigated the binding of specific DNA repair proteins via immunoflourescence. While manganese did not seem to induce DNA DSB and had no impact on NHEJ and MMEJ, HR and SSA were inhibited. In the case of chromate, the induction of DSB was further supported. Regarding DSB repair, no inhibition was seen in the case of NHEJ and SSA, but HR was diminished and MMEJ was activated in a pronounced manner. The results indicate a specific inhibition of error-free HR by manganese and chromate, with a shift towards error-prone DSB repair mechanisms in both cases. These observations suggest the induction of genomic instability and may explain the microsatellite instability involved in chromate-induced carcinogenicity.
| S-EPMC10298927 | biostudies-literature
Project description:The balance between major DNA double-strand break (DSB) repair pathways is influenced by binding of the Ku complex, a XRCC5/6 heterodimer, to DSB ends, initiating non-homologous end joining (NHEJ) but preventing additional DSB end resection and homologous recombination (HR). However, the key molecular cue for Ku recruitment to DSB sites is unknown. Here, we report that FOXL2, a forkhead family transcriptional factor, directs DSB repair pathway choice by acetylation-dependent binding to Ku. Upon DSB induction, SIRT1 translocates to the nucleus and deacetylates FOXL2 at lysine 124, leading to liberation of XRCC5 and XRCC6 from FOXL2 and formation of the Ku complex. FOXL2 ablation enhances Ku recruitment to DSB sites, imbalances DSB repair kinetics by accelerating NHEJ and inhibiting HR, and thus leads to catastrophic genomic events. Our study unveils the SIRT1-(de)acetylated FOXL2-Ku axis that governs the balance of DSB repair pathways to maintain genome integrity.
| S-EPMC7181608 | biostudies-literature
Project description:DNA nicks are the most common form of DNA damage, and if unrepaired can give rise to genomic instability. In human cells, nicks are efficiently repaired via the single-strand break repair pathway, but relatively little is known about the fate of nicks not processed by that pathway. Here we show that homology-directed repair (HDR) at nicks occurs via a mechanism distinct from HDR at double-strand breaks (DSBs). HDR at nicks, but not DSBs, is associated with transcription and is eightfold more efficient at a nick on the transcribed strand than at a nick on the nontranscribed strand. HDR at nicks can proceed by a pathway dependent upon canonical HDR factors RAD51 and BRCA2; or by an efficient alternative pathway that uses either ssDNA or nicked dsDNA donors and that is strongly inhibited by RAD51 and BRCA2. Nicks generated by either I-AniI or the CRISPR/Cas9(D10A) nickase are repaired by the alternative HDR pathway with little accompanying mutagenic end-joining, so this pathway may be usefully applied to genome engineering. These results suggest that alternative HDR at nicks may be stimulated in physiological contexts in which canonical RAD51/BRCA2-dependent HDR is compromised or down-regulated, which occurs frequently in tumors.
| S-EPMC3956201 | biostudies-other
Project description:FK506-binding proteins (FKBPs) alter the conformation of proteins via cis-trans isomerization of prolyl-peptide bonds. While this activity can be demonstrated in vitro, the intractability of detecting prolyl isomerization events in cells has limited our understanding of the biological processes regulated by FKBPs. Here we report that FKBP25 is an active participant in the repair of DNA double-strand breaks (DSBs). FKBP25 influences DSB repair pathway choice by promoting homologous recombination (HR) and suppressing single-strand annealing (SSA). Consistent with this observation, cells depleted of FKBP25 form fewer Rad51 repair foci in response to etoposide and ionizing radiation, and they are reliant on the SSA repair factor Rad52 for viability. We find that FKBP25's catalytic activity is required for promoting DNA repair, which is the first description of a biological function for this enzyme activity. Consistent with the importance of the FKBP catalytic site in HR, rapamycin treatment also impairs homologous recombination, and this effect is at least in part independent of mTor. Taken together these results identify FKBP25 as a component of the DNA DSB repair pathway.
| S-EPMC7457334 | biostudies-literature