Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It is well established that DNA damage induces checkpoint-mediated interphase arrest in higher eukaryotes, but recent studies demonstrate that DNA damage delays entry into anaphase as well. Damaged DNA in syncytial and gastrulating Drosophila embryos delays the metaphase/anaphase transition . In human cultured cells, DNA damage also induces a delay in mitosis . However, the mechanism by which DNA damage delays the anaphase onset is controversial. Some studies implicate a DNA damage checkpoint , whereas other studies invoke a spindle checkpoint . To resolve this issue, we compared the effects of random DNA breaks induced by X-irradiation to site-specific I-CreI endonuclease-induced chromosome breaks on cell-cycle progression in wild-type and checkpoint-defective Drosophila neuroblasts. We found that both the BubR1 spindle checkpoint pathway and the Grp/Chk1 DNA damage checkpoint pathway are involved in delaying the metaphase/anaphase transition after extensive X-irradiation-induced DNA damage, whereas Grp/Chk1, but not BubR1, is required to delay anaphase onset in the presence of I-CreI-induced double-strand breaks. On the basis of these results, we propose that DNA damage in nonkinetochore regions produces a Grp/Chk1 DNA-damage-checkpoint-mediated delay in the metaphase/anaphase transition.
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PMID:The Drosophila Grp/Chk1 DNA damage checkpoint controls entry into anaphase. 1572 94

The replication checkpoint kinase Cds1 preserves genome integrity by stabilizing stalled replication forks. Cds1 targets substrates through its FHA domain. The Cds1 FHA domain interacts with Mus81, a subunit of the Mus81-Eme1 structure-specific endonuclease. We report here that Mus81 and Rhp51 are required for generating deletion mutations in fission yeast replication mutants that experience replication stress. A mutation in the Mus81 FHA-binding motif eliminates its Cds1-binding and Cds1-dependent phosphorylation. Furthermore, this mutation exacerbates the deletion mutator phenotype of a replication mutant, and induces a hyper-recombination phenotype in hydroxyurea-treated cells. In unperturbed cells, Mus81 associates with chromatin throughout S phase. In replication mutants grown at semipermissive temperature, Mus81 undergoes minor Cds1-dependent phosphorylation, remains chromatin-associated, generates deletion mutations, and maintains cell growth. Upon S-phase arrest by acute hydroxyurea treatment, Mus81 is not required for cell viability but is essential for recovery from replication fork collapse. Moreover, Mus81 undergoes extensive Cds1-dependent phosphorylation and dissociates from chromatin in hydroxyurea-arrested cells, thereby preventing it from cleaving stalled replication forks that could lead to fork breakage and chromosomal rearrangement. These results provide novel insights into how Cds1 regulates Mus81 accordingly when cells experience different replication stress to preserve genome integrity.
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PMID:Replication checkpoint kinase Cds1 regulates Mus81 to preserve genome integrity during replication stress. 1580 65

Members of the RecQ family of DNA helicases, mutated in several syndromes associated with cancer predisposition, are key regulators of genome stability. The Saccharomyces cerevisiae SLX4 gene is required for cell viability in the absence of Sgs1, the only yeast RecQ helicase. SLX4 encodes one subunit of the heterodimeric Slx1-Slx4 endonuclease, although its cellular function is not clear. Slx1-Slx4 was reported to preferentially cleave replication fork-like structures in vitro, and cells lacking SLX4 are hypersensitive to DNA alkylation damage. Here we report that Slx4 becomes phosphorylated in cells exposed to a wide range of genotoxins. Even though it has been proposed that the role of Slx4 is restricted to S-phase, Slx4 phosphorylation is observed in cells arrested in G1 or G2 phases of the cell cycle, but not during an unperturbed cell cycle. Slx4 phosphorylation is completely abolished in cells lacking the Mec1 and Tel1 protein kinases, critical regulators of genome stability, but is barely affected in the absence of both Rad53 and Chk1 kinases. Finally we show that, whereas both Slx1 and Slx4 are dispensable for activation of cell-cycle checkpoints, Slx4, but not Slx1, is required for repair of DNA alkylation damage in both aynchronously growing cells and in G2-phase-arrested cells. These results reveal Slx4 as a new target of the Mec1/Tel1 kinases, with a crucial role in DNA repair that is not restricted to the processing of stalled replisomes.
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PMID:Slx4 becomes phosphorylated after DNA damage in a Mec1/Tel1-dependent manner and is required for repair of DNA alkylation damage. 1597 89

The Mus81-Eme1 complex is a structure-specific endonuclease that preferentially cleaves nicked Holliday junctions, 3'-flap structures and aberrant replication fork structures. Mus81-/- mice have been shown to exhibit spontaneous chromosomal aberrations and, in one of two models, a predisposition to cancers. The molecular mechanisms underlying its role in chromosome integrity, however, are largely unknown. To clarify the role of Mus81 in human cells, we deleted the gene in the human colon cancer cell line HCT116 by gene targeting. Here we demonstrate that Mus81 confers resistance to DNA crosslinking agents and slight resistance to other DNA-damaging agents. Mus81 deficiency spontaneously promotes chromosome damage such as breaks and activates the intra-S-phase checkpoint through the ATM-Chk1/Chk2 pathways. Furthermore, Mus81 deficiency activates the G2/M checkpoint through the ATM-Chk2 pathway and promotes DNA rereplication. Increased rereplication is reversed by the ectopic expression of Cdk1. Haploinsufficiency of Mus81 or Eme1 also causes similar phenotypes. These findings suggest that a complex network of the checkpoint pathways that respond to DNA double-strand breaks may participate in some of the phenotypes associated with Mus81 or Eme1 deficiency.
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PMID:Haploinsufficiency of the Mus81-Eme1 endonuclease activates the intra-S-phase and G2/M checkpoints and promotes rereplication in human cells. 1645 34

Mutations in genome caretaker genes can induce genomic instability, which are potentially early events in tumorigenesis. Cells have evolved biological processes to cope with the genomic insults. One is a multifaceted response, termed checkpoint, which is a network of signaling pathways to coordinate cell cycle transition with DNA repair, activation of transcriptional programs, and induction of tolerance of the genomic perturbations. When genomic perturbations are beyond repair, checkpoint responses can also induce apoptosis or senescence to eliminate those deleterious damaged cells. Fission yeast, Schizosaccharomyces pombe (S. pombe) has served as a valuable model organism for studies of the checkpoint signaling pathways. In this chapter, we describe methods used to analyze mutagenesis and recombinational repair induced by genomic perturbations, and methods used to detect the checkpoint responses to replication stress and DNA damage in fission yeast cells. In the first section, we present methods used to analyze the mutation rate, mutation spectra, and recombinational repair in fission yeast when replication is perturbed by either genotoxic agents or mutations in genomic caretaker gene such as DNA replication genes. In the second section, we describe methods used to examine checkpoint activation in response to chromosome replication stress and DNA damage. In the final section, we comment on how checkpoint activation regulates mutagenic synthesis by a translesion DNA polymerase in generating a mutator phenotype of small sequence alterations in cells, and how a checkpoint kinase appropriately regulates an endonuclease complex to either prevent or allow deletion of genomic sequences and recombinational repair when fission yeast cells experience genomic perturbation in order to avoid deleterious mutations and maintain cell growth.
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PMID:Methods for studying mutagenesis and checkpoints in Schizosaccharomyces pombe. 1679 2

Previous studies in yeast have suggested that the SGS1 DNA helicase or the Mus81-Mms4 structure-specific endonuclease is required to suppress the accumulation of lethal recombination intermediates during DNA replication. However, the structure of these intermediates and their mechanism of the suppression are unknown. To examine this reaction, we have isolated and characterized a temperature-sensitive (ts) allele of MUS81. At the non-permissive temperature, sgs1Deltamus81(ts) cells arrest at G(2)/M phase after going through S-phase. Bulk DNA replication appears complete but is defective since the Rad53 checkpoint kinase is strongly phosphorylated under these conditions. In addition, the induction of Rad53 hyper-phosphorylation by MMS was deficient at permissive temperature. Analysis of rDNA replication intermediates at the non-permissive temperature revealed elevated pausing of replication forks at the RFB in the sgs1Deltamus81(ts) mutant and a novel linear structure that was dependent on RAD52. Pulsed-field gel electrophoresis of the mus81Delta mutant revealed an expansion of the rDNA locus depending on RAD52, in addition to fragmentation of Chr XII in the sgs1Deltamus81(ts) mutant at permissive temperature. This is the first evidence that Mus81 functions in quality control of replication forks and that it is involved in the maintenance of rDNA repeats in vivo.
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PMID:Mus81 functions in the quality control of replication forks at the rDNA and is involved in the maintenance of rDNA repeat number in Saccharomyces cerevisiae. 1755 73

A single HO endonuclease-induced double-strand break (DSB) is sufficient to activate the DNA damage checkpoint and cause Saccharomyces cells to arrest at G(2)/M for 12-14 h, after which cells adapt to the presence of the DSB and resume cell cycle progression. The checkpoint signal leading to G(2)/M arrest was previously shown to be nuclear-limited. Cells lacking ATR-like Mec1 exhibit no DSB-induced cell cycle delay; however, cells lacking Mec1's downstream protein kinase targets, Rad53 or Chk1, still have substantial G(2)/M delay, as do cells lacking securin, Pds1. This delay is eliminated only in the triple mutant chk1Delta rad53Delta pds1Delta, suggesting that Rad53 and Chk1 control targets other than the stability of securin in enforcing checkpoint-mediated cell cycle arrest. The G(2)/M arrest in rad53Delta and chk1Delta revealed a unique cytoplasmic phenotype in which there are frequent dynein-dependent excursions of the nucleus through the bud neck, without entering anaphase. Such excursions are infrequent in wild-type arrested cells, but have been observed in cells defective in mitotic exit, including the semidominant cdc5-ad mutation. We suggest that Mec1-dependent checkpoint signaling through Rad53 and Chk1 includes the repression of nuclear movements that are normally associated with the execution of anaphase.
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PMID:The yeast DNA damage checkpoint proteins control a cytoplasmic response to DNA damage. 1758 85

Budding yeast Mec1, encoded by the yeast ATR/ATM homolog, negatively regulates cell cycle progression by activating Rad53 (Chk2) and Chk1, two parallel downstream checkpoint pathways. Chk1 phosphorylates Pds1 (securin), which prevents Pds1 degradation. We determined whether activation of both downstream pathways is required to establish G2 arrest in response to double-strand breaks (DSBs). In a hypomorphic mec1 mutant, Rad53 activation was not required to establish G2 arrest triggered by a single HO endonuclease-generated DSB. However, Pds1 phosphorylation did correlate with G2 arrest and mec1-21 pds1 cells did not arrest in G2 after exposure to ionizing radiation. The G2 checkpoint genes, CHK1 and PDS1, did confer radiation resistance in mec1-21, indicating that CHK1-mediated pathway is functional in the mec1 hypomorph. Thus, phosphorylation of Pds1 but not Rad53 correlates with G2 arrest in response to DSBs in the mec1 hypomorphic mutant.
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PMID:Activation of the budding yeast securin Pds1 but not Rad53 correlates with double-strand break-associated G2/M cell cycle arrest in a mec1 hypomorphic mutant. 1767 32

The mammalian ERCC1-XPF endonuclease has a suggested role in the repair of DNA double-strand breaks (DSB) by single-strand annealing (SSA). Here, we investigated the role of ERCC1 in homologous recombination in mammalian cells, and confirm a role of ERCC1 in SSA. Interestingly, we also report an unexpected role for ERCC1 in gene conversion. This provides support that gene conversion in mammalian somatic cells is carried out through synthesis-dependent strand annealing, rather than through a double Holliday Junction mechanism. Moreover, we find low frequencies of SSA and gene conversion in G1-arrested cells, suggesting that SSA is not a frequent DSB repair pathway in G1-arrested mammalian cells, even in the presence of perfect repeats. Furthermore, we find that SSA is not influenced by inhibition of CDK2 (using Roscovitine), ATM (using Caffeine and KU55933), Chk1 (using CEP-3891) or DNA-PK (using NU7026).
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PMID:The ERCC1/XPF endonuclease is required for efficient single-strand annealing and gene conversion in mammalian cells. 1796 1

During replication arrest, the DNA replication checkpoint plays a crucial role in the stabilization of the replisome at stalled forks, thus preventing the collapse of active forks and the formation of aberrant DNA structures. How this checkpoint acts to preserve the integrity of replication structures at stalled fork is poorly understood. In Schizosaccharomyces pombe, the DNA replication checkpoint kinase Cds1 negatively regulates the structure-specific endonuclease Mus81/Eme1 to preserve genomic integrity when replication is perturbed. Here, we report that, in response to hydroxyurea (HU) treatment, the replication checkpoint prevents S-phase-specific DNA breakage resulting from Mus81 nuclease activity. However, loss of Mus81 regulation by Cds1 is not sufficient to produce HU-induced DNA breaks. Our results suggest that unscheduled cleavage of stalled forks by Mus81 is permitted when the replisome is not stabilized by the replication checkpoint. We also show that HU-induced DNA breaks are partially dependent on the Rqh1 helicase, the fission yeast homologue of BLM, but are independent of its helicase activity. This suggests that efficient cleavage of stalled forks by Mus81 requires Rqh1. Finally, we identified an interplay between Mus81 activity at stalled forks and the Chk1-dependent DNA damage checkpoint during S-phase when replication forks have collapsed.
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PMID:Cleavage of stalled forks by fission yeast Mus81/Eme1 in absence of DNA replication checkpoint. 1803 83


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