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)

RAD52 and RAD9 are required for the repair of double-strand breaks (DSBs) induced by physical and chemical DNA-damaging agents in Saccharomyces cerevisiae. Analysis of EcoRI endonuclease expression in vivo revealed that, in contrast to DSBs containing damaged or modified termini, chromosomal DSBs retaining complementary ends could be repaired in rad52 mutants and in G1-phase Rad+ cells. Continuous EcoRI-induced scission of chromosomal DNA blocked the growth of rad52 mutants, with most cells arrested in G2 phase. Surprisingly, rad52 mutants were not more sensitive to EcoRI-induced cell killing than wild-type strains. In contrast, endonuclease expression was lethal in cells deficient in Ku-mediated end joining. Checkpoint-defective rad9 mutants did not arrest cell cycling and lost viability rapidly when EcoRI was expressed. Synthesis of the endonuclease produced extensive breakage of nuclear DNA and stimulated interchromosomal recombination. These results and those of additional experiments indicate that cohesive ended DSBs in chromosomal DNA can be accurately repaired by RAD52-mediated recombination and by recombination-independent complementary end joining in yeast cells.
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PMID:Requirement for end-joining and checkpoint functions, but not RAD52-mediated recombination, after EcoRI endonuclease cleavage of Saccharomyces cerevisiae DNA. 952 60

In yeast, broken chromosomes can be repaired by recombination, resulting in nonreciprocal translocations. In haploid cells suffering an HO endonuclease-induced, double-strand break (DSB), nearly 2% of the broken chromosome ends recombined with a sequence near the opposite chromosome end, which shares only 72 bp of homology with the cut sequence. This produced a repaired chromosome with the same 20-kb sequence at each end. Diploid strains were constructed in which the broken chromosome shared homology with the unbroken chromosome only on the centromere-proximal side of the DSB. More than half of these cells repaired the DSB by copying sequences distal to the break from the unbroken template chromosome. All these events were RAD52 dependent. Pedigree analysis established that DSBs occurring in G1 were repaired by a replicative mechanism, producing two identical daughter cells. We discuss the implications of these data in understanding telomerase-independent replication of telomeres, gene amplification, and the evolution of chromosomal ends.
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PMID:Chromosome break-induced DNA replication leads to nonreciprocal translocations and telomere capture. 979 56

Homothallic Saccharomyces cerevisiae strains switch their mating-type in a specific gene conversion event induced by a DNA double strand break made by the HO endonuclease. The RAD52 group genes control recombinational repair of DNA double strand breaks, and we examined their role in native homothallic mating-type switching. Surprisingly, we found that the Rad54 protein was important but not essential for mating-type switching under natural conditions. As an upper limit, we estimate that 29% of the rad54 spore clones can successfully switch their mating-type. The RAD55 and RAD57 gene products were even less important, but their presence increased the efficiency of the process. In contrast, the RAD51 and RAD52 genes are essential for homothallic mating-type switching. We propose that mating-type switching in RAD54 mutants occurs stochastically with a low probability, possibly reflecting different states of chromosomal structure.
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PMID:The Saccharomyces cerevisiae RAD54 gene is important but not essential for natural homothallic mating-type switching. 992 34

The removal of oxidative damage from Saccharomyces cerevisiae DNA is thought to be conducted primarily through the base excision repair pathway. The Escherichia coli endonuclease III homologs Ntg1p and Ntg2p are S. cerevisiae N-glycosylase-associated apurinic/apyrimidinic (AP) lyases that recognize a wide variety of damaged pyrimidines (H. J. You, R. L. Swanson, and P. W. Doetsch, Biochemistry 37:6033-6040, 1998). The biological relevance of the N-glycosylase-associated AP lyase activity in the repair of abasic sites is not well understood, and the majority of AP sites in vivo are thought to be processed by Apn1p, the major AP endonuclease in yeast. We have found that yeast cells simultaneously lacking Ntg1p, Ntg2p, and Apn1p are hyperrecombinogenic (hyper-rec) and exhibit a mutator phenotype but are not sensitive to the oxidizing agents H2O2 and menadione. The additional disruption of the RAD52 gene in the ntg1 ntg2 apn1 triple mutant confers a high degree of sensitivity to these agents. The hyper-rec and mutator phenotypes of the ntg1 ntg2 apn1 triple mutant are further enhanced by the elimination of the nucleotide excision repair pathway. In addition, removal of either the lesion bypass (Rev3p-dependent) or recombination (Rad52p-dependent) pathway specifically enhances the hyper-rec or mutator phenotype, respectively. These data suggest that multiple pathways with overlapping specificities are involved in the removal of, or tolerance to, spontaneous DNA damage in S. cerevisiae. In addition, the fact that these responses to induced and spontaneous damage depend upon the simultaneous loss of Ntg1p, Ntg2p, and Apn1p suggests a physiological role for the AP lyase activity of Ntg1p and Ntg2p in vivo.
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PMID:Overlapping specificities of base excision repair, nucleotide excision repair, recombination, and translesion synthesis pathways for DNA base damage in Saccharomyces cerevisiae. 1008 60

A DNA double-strand break (DSB) created by the HO endonuclease in Saccharomyces cerevisiae will stimulate recombination between flanking repeats by the single-strand annealing (SSA) pathway, producing a deletion. Previously the efficiency of SSA, using homologous sequences of different lengths, was measured in competition with that of a larger repeat further from the DSB, which ensured that nearly all cells would survive the DSB if the smaller region was not used (N. Sugawara and J. E. Haber, Mol. Cell. Biol. 12:563-575, 1992). Without competition, the efficiency with which homologous segments of 63 to 205 bp engaged in SSA was significantly increased. A sequence as small as 29 bp was used 0.2% of the time, and homology dependence was approximately linear up to 415 bp, at which size almost all cells survived. A mutant with a deletion of RAD59, a homologue of RAD52, was defective for SSA, especially when the homologous-sequence length was short; however, even with 1.17-kb substrates, SSA was reduced fourfold. DSB-induced gene conversion also showed a partial dependence on Rad59p, again being greatest when the homologous-sequence length was short. We found that Rad59p plays a role in removing nonhomologous sequences from the ends of single-stranded DNA when it invades a homologous DNA template, in a manner similar to that previously seen with srs2 mutants. Deltarad59 affected DSB-induced gene conversion differently from msh3 and msh2, which are also defective in removing nonhomologous ends in both DSB-induced gene conversion and SSA. A msh3 rad59 double mutant was more severely defective in SSA than either single mutant.
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PMID:DNA length dependence of the single-strand annealing pathway and the role of Saccharomyces cerevisiae RAD59 in double-strand break repair. 1086 86

Possible functions of previously described genes RAD29 and RAD31 involved in DNA repair were determined by analyzing the interaction between these genes and mutations in the genes of the three basic epistatic groups: RAD3 (nucleotide excision repair), RAD6 (error-prone mutagenic repair system), RAD52 (recombination repair pathway), and also the apn1 mutation that blocks the synthesis of major AP endonuclease (base excision repair). The results obtained in these studies and the estimation of the capability for excision repair of lesions induced by 8-metoxipsoralen and subsequent exposure to long-wavelength UV light in mutants for these genes led to the assumption that the RAD29 and RAD31 genes are involved in yeast DNA repair control.
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PMID:[RAD29 and RAD31--new genes from Saccharomyces cerevisiae yeasts, participating in control of DNA repair. II. Clarification of possible functions of these genes]. 1103 72

Broken chromosomes can be repaired by several homologous recombination mechanisms, including gene conversion and break-induced replication (BIR). In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) is normally repaired by gene conversion. Previously, we have shown that in the absence of RAD52, repair is nearly absent and diploid cells lose the broken chromosome; however, in cells lacking RAD51, gene conversion is absent but cells can repair the DSB by BIR. We now report that gene conversion is also abolished when RAD54, RAD55, and RAD57 are deleted but BIR occurs, as with rad51Delta cells. DSB-induced gene conversion is not significantly affected when RAD50, RAD59, TID1 (RDH54), SRS2, or SGS1 is deleted. Various double mutations largely eliminate both gene conversion and BIR, including rad51Delta rad50Delta, rad51Delta rad59Delta, and rad54Delta tid1Delta. These results demonstrate that there is a RAD51- and RAD54-independent BIR pathway that requires RAD59, TID1, RAD50, and presumably MRE11 and XRS2. The similar genetic requirements for BIR and telomere maintenance in the absence of telomerase also suggest that these two processes proceed by similar mechanisms.
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PMID:Genetic requirements for RAD51- and RAD54-independent break-induced replication repair of a chromosomal double-strand break. 1123 40

Cells of vertebrates remove DNA double-strand breaks (DSBs) from their genome predominantly utilizing a fast, DNA-PKcs-dependent form of non-homologous end joining (D-NHEJ). Mutants with inactive DNA-PKcs remove the majority of DNA DSBs utilizing a slow, DNA-PKcs-independent pathway that does not utilize genes of the RAD52 epistasis group, is error-prone and can therefore be classified as a form of NHEJ (termed basic or B-NHEJ). We studied the role of DNA ligase IV in these pathways of NHEJ. Although biochemical studies show physical and functional interactions between the DNA-PKcs/Ku and the DNA ligase IV/Xrcc4 complexes suggesting operation within the same pathway, genetic evidence to support this notion is lacking in mammalian cells. Primary human fibroblasts (180BR) with an inactivating mutation in DNA ligase IV, rejoined DNA DSBs predominantly with slow kinetics similar to those observed in cells deficient in DNA-PKcs, or in wild-type cells treated with wortmannin to inactivate DNA-PK. Treatment of 180BR cells with wortmannin had only a small effect on DNA DSB rejoining and no effect on cell radiosensitivity to killing although it sensitized control cells to 180BR levels. This is consistent with DNA ligase IV functioning as a component of the D-NHEJ, and demonstrates the unperturbed operation of the DNA-PKcs-independent pathway (B-NHEJ) at significantly reduced levels of DNA ligase IV. In vitro, extracts of 180BR cells supported end joining of restriction endonuclease-digested plasmid to the same degree as extracts of control cells when tested at 10 mM Mg(2+). At 0.5 mM Mg(2+), where only DNA ligase IV is expected to retain activity, low levels of end joining ( approximately 10% of 10 mM) were seen in the control but there was no detectable activity in 180BR cells. Antibodies raised against DNA ligase IV did not measurably inhibit end joining at 10 mM Mg(2+) in either cell line. Thus, in contrast to the situation in vivo, end joining in vitro is dominated by pathways with properties similar to B-NHEJ that do not display a strong dependence on DNA ligase IV, with D-NHEJ retaining only a limited contribution. The implications of these observations to studies of NHEJ in vivo and in vitro are discussed.
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PMID:Genetic evidence for the involvement of DNA ligase IV in the DNA-PK-dependent pathway of non-homologous end joining in mammalian cells. 1129 37

Many DNA polymerases (Pol) have an intrinsic 3'-->5' exonuclease (Exo) activity which corrects polymerase errors and prevents mutations. We describe a role of the 3'-->5' Exo of Pol delta as a supplement or backup for the Rad27/Fen1 5' flap endonuclease. A yeast rad27 null allele was lethal in combination with Pol delta mutations in Exo I, Exo II, and Exo III motifs that inactivate its exonuclease, but it was viable with mutations in other parts of Pol delta. The rad27-p allele, which has little phenotypic effect by itself, was also lethal in combination with mutations in the Pol delta Exo I and Exo II motifs. However, rad27-p Pol delta Exo III double mutants were viable. They exhibited strong synergistic increases in CAN1 duplication mutations, intrachromosomal and interchromosomal recombination, and required the wild-type double-strand break repair genes RAD50, RAD51, and RAD52 for viability. Observed effects were similar to those of the rad27-null mutant deficient in the removal of 5' flaps in the lagging strand. These results suggest that the 3'-->5' Exo activity of Pol delta is redundant with Rad27/Fen1 for creating ligatable nicks between adjacent Okazaki fragments, possibly by reducing the amount of strand-displacement in the lagging strand.
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PMID:The 3'-->5' exonuclease of DNA polymerase delta can substitute for the 5' flap endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome instability. 1130 2

Cells can repair DNA double-strand breaks by both homologous and nonhomologous mechanisms. To explore the basis of pathway utilization, we developed both plasmid and chromosomal yeast repair assays in which breaks are created with restriction endonucleases so that nonhomologous end-joining (NHEJ) competes with the single-strand annealing (SSA) recombination pathway, which we show acts with high efficiency via terminal direct repeats of only 28 bp and with reduced but measurable efficiency at 10 bp. The chromosomal assay utilizes a novel approach termed suicide deletion in which the endonuclease cleaves its own gene from the chromosome, thereby ending the futile cleavage cycle that otherwise prevents detection of simple-religation events. Eliminating SSA as a possibility in either assay, either by removal of the direct repeat or by mutation of RAD52, increased the relative but not the absolute efficiency of NHEJ. In contrast, the apparent efficiency of NHEJ was specifically increased in the G1 stage of the haploid cell cycle, as well as by the glucose depletion-signaled transition to stationary phase. The combined results argue against a model in which pathway utilization is determined by a passive competition. Instead, they demonstrate an active regulation designed to optimize the likelihood of genome restoration based on cell state.
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PMID:Enhancement of Saccharomyces cerevisiae end-joining efficiency by cell growth stage but not by impairment of recombination. 1213 7


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