Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair.
Ontology highlight
ABSTRACT:
SUBMITTER: Jamsen JA
PROVIDER: S-EPMC8379156 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Oxidized dGTP (8-oxo-7,8-dihydro-2´-deoxyguanosine triphosphate, 8-oxodGTP) insertion by DNA polymerases strongly promotes cancer and human disease. How DNA polymerases discriminate against oxidized and undamaged nucleotides, especially in error-prone double strand break (DSB) repair, is poorly understood. High-resolution time-lapse X-ray crystallography snapshots of DSB repair polymerase μ undergoing DNA synthesis reveal that a third active site metal promotes insertion of oxidized and undamaged dGTP in the canonical anti-conformation opposite template cytosine. The product metal bridged O8 with product oxygens, and was not observed in the syn-conformation opposite template adenine (At). Rotation of At into the syn-conformation enabled undamaged dGTP misinsertion. Exploiting metal and substrate dynamics in a rigid active site allows 8-oxodGTP to circumvent polymerase fidelity safeguards to promote pro-mutagenic double strand break repair.
| S-EPMC8024293 | biostudies-literature
Project description:Pharmacologic inhibitors of poly(ADP-ribose) polymerase (PARP) putatively enhance radiation toxicity in cancer cells. Although there is considerable information on the molecular interactions of PARP and BRCA1- and BRCA2-deficient cancers, very little is known of the PARP inhibition effect upon cancers proficient in DNA double-strand break repair after ionizing radiation or after stalled replication forks. In this work, we investigate whether PARP inhibition by ABT-888 (veliparib) augments death-provoking effects of ionizing radiation, or of the topoisomerase I poison topotecan, within uterine cervix cancers cells harboring an unfettered, overactive ribonucleotide reductase facilitating DNA double-strand break repair and contrast these findings with ovarian cancer cells whose regulation of ribonucleotide reductase is relatively intact. Cell lethality of a radiation-ABT-888 combination is radiation and drug dose dependent. Data particularly highlight an enhanced topotecan-ABT-888 cytotoxicity, and corresponds to an increased number of unrepaired DNA double-strand breaks. Overall, our findings support enhanced radiochemotherapy toxicity in cancers proficient in DNA double-strand break repair when PARP is inhibited by ABT-888.
| S-EPMC3588069 | biostudies-literature
Project description:The yeast Chk2/Chk1 homolog Rad53 is a central component of the DNA damage checkpoint system. While it controls genotoxic stress responses such as cell cycle arrest, replication fork stabilization and increase in dNTP pools, little is known about the consequences of reduced Rad53 levels on the various cellular endpoints or about its roles in dealing with chronic vs. acute genotoxic challenges. Using a tetraploid gene dosage model in which only one copy of the yeast RAD53 is functional (simplex), we found that the simplex strain was not sensitive to acute UV radiation or chronic MMS exposure. However, the simplex strain was sensitized to chronic exposure of the ribonucleotide reductase inhibitor hydroxyurea (HU). Surprisingly, reduced RAD53 gene dosage did not affect sensitivity to HU acute exposure, indicating that immediate checkpoint responses and recovery from HU-induced stress were not compromised. Interestingly, cells of most of the colonies that arise after chronic HU exposure acquired heritable resistance to HU. We also found that short HU exposure before and after treatment of G? cells with ionizing radiation (IR) reduced the capability of RAD53 simplex cells to repair DSBs, in agreement with sensitivity of RAD53 simplex strain to high doses of IR. We propose that a modest reduction in Rad53 activity can impact the activation of the ribonucleotide reductase catalytic subunit Rnr1 following stress, reducing the ability to generate nucleotide pools sufficient for DNA repair and replication. At the same time, reduced Rad53 activity may lead to genome instability and to the acquisition of drug resistance before and/or during the chronic exposure to HU. These results have implications for developing drug enhancers as well as for understanding mechanisms of drug resistance in cells compromised for DNA damage checkpoint.
| S-EPMC3631316 | biostudies-literature
Project description:Shieldin complex subunit 3 (SHLD3) is the apical subunit of a recently-identified shieldin complex and plays a critical role in DNA double-strand break repair. To fulfill its function in DNA repair, SHLD3 interacts with the mitotic spindle assembly checkpoint protein REV7 homolog (REV7), but the details of this interaction remain obscure. Here, we present the crystal structures of REV7 in complex with SHLD3's REV7-binding domain (RBD) at 2.2-2.3 Å resolutions. The structures revealed that the ladle-shaped RBD in SHLD3 uses its N-terminal loop and C-terminal ?-helix (?C-helix) in its interaction with REV7. The N-terminal loop exhibited a structure similar to those previously identified in other REV7-binding proteins, and the less-conserved ?C-helix region adopted a distinct mode for binding REV7. In vitro and in vivo binding analyses revealed that the N-terminal loop and the ?C-helix are both indispensable for high-affinity REV7 binding (with low-nanomolar affinity), underscoring the crucial role of SHLD3 ?C-helix in protein binding. Moreover, binding kinetics analyses revealed that the REV7 "safety belt" region, which plays a role in binding other proteins, is essential for SHLD3-REV7 binding, as this region retards the dissociation of the RBD from the bound REV7. Together, the findings of our study reveal the molecular basis of the SHLD3-REV7 interaction and provide critical insights into how SHLD3 recognizes REV7.
| S-EPMC6952594 | biostudies-literature
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
Project description:Hereditary mutations in polynucleotide kinase-phosphatase (PNKP) result in a spectrum of neurological pathologies ranging from neurodevelopmental dysfunction in microcephaly with early onset seizures (MCSZ) to neurodegeneration in ataxia oculomotor apraxia-4 (AOA4) and Charcot-Marie-Tooth disease (CMT2B2). Consistent with this, PNKP is implicated in the repair of both DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs); lesions that can trigger neurodegeneration and neurodevelopmental dysfunction, respectively. Surprisingly, however, we did not detect a significant defect in DSB repair (DSBR) in primary fibroblasts from PNKP patients spanning the spectrum of PNKP-mutated pathologies. In contrast, the rate of SSB repair (SSBR) is markedly reduced. Moreover, we show that the restoration of SSBR in patient fibroblasts collectively requires both the DNA kinase and DNA phosphatase activities of PNKP, and the fork-head associated (FHA) domain that interacts with the SSBR protein, XRCC1. Notably, however, the two enzymatic activities of PNKP appear to affect different aspects of disease pathology, with reduced DNA phosphatase activity correlating with neurodevelopmental dysfunction and reduced DNA kinase activity correlating with neurodegeneration. In summary, these data implicate reduced rates of SSBR, not DSBR, as the source of both neurodevelopmental and neurodegenerative pathology in PNKP-mutated disease, and the extent and nature of this reduction as the primary determinant of disease severity.
| S-EPMC7337934 | biostudies-literature
Project description:Double-strand breaks (DSBs) are among the most deleterious lesions DNA can endure. Yet, DSBs are programmed at the onset of meiosis, and are required to facilitate appropriate reduction of ploidy in daughter cells. Repair of these breaks is tightly controlled to favor homologous recombination (HR)-the only repair pathway that can form crossovers. However, little is known about how the activities of alternative repair pathways are regulated at these stages. We discovered an unexpected synthetic interaction between the DSB machinery and strand-exchange proteins. Depleting the Caenorhabditis elegans DSB-promoting factors HIM-5 and DSB-2 suppresses the formation of chromosome fusions that arise in the absence of RAD-51 or other strand-exchange mediators. Our investigations reveal that nonhomologous and theta-mediated end joining (c-NHEJ and TMEJ, respectively) and single strand annealing (SSA) function redundantly to repair DSBs when HR is compromised, and that HIM-5 influences the utilization of TMEJ and SSA.
| S-EPMC6218235 | biostudies-literature
Project description:Double-strand DNA breaks occur upon exposure of cells to ionizing radiation and certain chemical agents or indirectly through replication fork collapse at DNA damage sites. If left unrepaired, double-strand breaks can cause genome instability and cell death, and their repair can result in loss of heterozygosity. In response to DNA damage, proteins involved in double-strand break repair by homologous recombination relocalize into discrete nuclear foci. We identified 29 proteins that colocalize with recombination repair protein Rad52 in response to DNA damage. Of particular interest, Ygr042w/Mte1, a protein of unknown function, showed robust colocalization with Rad52. Mte1 foci fail to form when the DNA helicase gene MPH1 is absent. Mte1 and Mph1 form a complex and are recruited to double-strand breaks in vivo in a mutually dependent manner. MTE1 is important for resolution of Rad52 foci during double-strand break repair and for suppressing break-induced replication. Together our data indicate that Mte1 functions with Mph1 in double-strand break repair.
| S-EPMC4858770 | biostudies-literature
Project description:c-Myc is a transcriptional factor that functions as a central regulator of cell growth, proliferation, and apoptosis. Overexpression of c-Myc also enhances DNA double-strand breaks (DSBs), genetic instability, and tumorigenesis. However, the mechanism(s) involved remains elusive. Here, we discovered that ?-ray ionizing radiation-induced DSBs promote c-Myc to form foci and to co-localize with ?-H2AX. Conditional expression of c-Myc in HO15.19 c-Myc null cells using the Tet-Off/Tet-On inducible system results in down-regulation of Ku DNA binding and suppressed activities of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and DNA end-joining, leading to inhibition of DSB repair and enhanced chromosomal and chromatid breaks. Expression of c-Myc reduces both signal and coding joins with decreased fidelity during V(D)J recombination. Mechanistically, c-Myc directly interacts with Ku70 protein through its Myc box II (MBII) domain. Removal of the MBII domain from c-Myc abrogates its inhibitory effects on Ku DNA binding, DNA-PKcs, and DNA end-joining activities, which results in loss of c-Myc's ability to block DSB repair and V(D)J recombination. Interestingly, c-Myc directly disrupts the Ku/DNA-PKcs complex in vitro and in vivo. Thus, c-Myc suppression of DSB repair and V(D)J recombination may occur through inhibition of the nonhomologous end-joining pathway, which provides insight into the mechanism of c-Myc in the development of tumors through promotion of genomic instability.
| S-EPMC3540944 | biostudies-literature
Project description:ERCC1-XPF endonuclease is required for nucleotide excision repair (NER) of helix-distorting DNA lesions. However, mutations in ERCC1 or XPF in humans or mice cause a more severe phenotype than absence of NER, prompting a search for novel repair activities of the nuclease. In Saccharomyces cerevisiae, orthologs of ERCC1-XPF (Rad10-Rad1) participate in the repair of double-strand breaks (DSBs). Rad10-Rad1 contributes to two error-prone DSB repair pathways: microhomology-mediated end joining (a Ku86-independent mechanism) and single-strand annealing. To determine if ERCC1-XPF participates in DSB repair in mammals, mutant cells and mice were screened for sensitivity to gamma irradiation. ERCC1-XPF-deficient fibroblasts were hypersensitive to gamma irradiation, and gammaH2AX foci, a marker of DSBs, persisted in irradiated mutant cells, consistent with a defect in DSB repair. Mutant mice were also hypersensitive to irradiation, establishing an essential role for ERCC1-XPF in protecting against DSBs in vivo. Mice defective in both ERCC1-XPF and Ku86 were not viable. However, Ercc1(-/-) Ku86(-/-) fibroblasts were hypersensitive to gamma irradiation compared to single mutants and accumulated significantly greater chromosomal aberrations. Finally, in vitro repair of DSBs with 3' overhangs led to large deletions in the absence of ERCC1-XPF. These data support the conclusion that, as in yeast, ERCC1-XPF facilitates DSB repair via an end-joining mechanism that is Ku86 independent.
| S-EPMC2519706 | biostudies-literature