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)

Abasic (AP) sites in DNA are potentially lethal and mutagenic. 'Class II' AP endonucleases initiate the repair of these and other DNA lesions. In yeast, the predominant enzyme of this type is Apn1, and its elimination sensitizes the cells to killing by simple alkylating agents or oxidants, and raises the rate of spontaneous mutation. We investigated the ability of the major human class II AP endonuclease, Ape, which is structurally unrelated to Apn1, to replace the yeast enzyme in vivo. Confocal immunomicroscopy studies indicate that approximately 25% of the Ape expressed in yeast is present in the nucleus. High-level Ape expression corresponding to approximately 7000 molecules per nucleus, equal to the normal Apn1 copy number, restored resistance to methyl methanesulfonate to near wild-type levels in Apn1-deficient (apn1-) yeast. Ape expression in apn1- yeast provided little protection against H2O2 challenges, consistent with the weak 3'-repair diesterase activity of the human enzyme. Ape expression at approximately 2000 molecules per nucleus reduced the spontaneous mutation rate of apn1- yeast to that seen for wild-type cells. Because Ape has a powerful AP endonuclease but weak 3'-diesterase activity, these findings indicate that endogenously generated AP sites can drive spontaneous mutagenesis.
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PMID:Trans-complementation by human apurinic endonuclease (Ape) of hypersensitivity to DNA damage and spontaneous mutator phenotype in apn1-yeast. 855 61

The multifunctional DNA repair enzyme (APEX nuclease) having apurinic/apyrimidinic (AP) endonuclease, 3'-5' exonuclease, DNA 3' repair diesterase and DNA 3'-phosphatase activities is thought to be involved in repair of AP sites and single-strand breaks with 3'-blocked termini. To investigate the biological role of the enzyme, we studied the correlation between APEX AP endonuclease activity in several human glioma cell lines having various degree of its expression and cellular susceptibility to cytotoxic agents such as methyl methanesulfonate (MMS), 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3- (2-chloroethyl)-3-nitrosourea hydrochloride (ACNU), cis-diamminedichloroplatinum(II) (CDDP), etoposide (VP-16), hydrogen peroxide (H2O2), hyperthermia and X-ray. The cell lines having lower APEX expression showed higher sensitivity to MMS and H2O2 which are known to induce AP sites and single strand breaks on DNA, respectively. The cellular susceptibility to the other agents tested was not significantly correlated to the APEX expression. The present results are thought to support the notion that APEX nuclease plays an important role on repair of AP sites and single-strand DNA breaks with 3'-blocked termini in mammalian cells.
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PMID:Relationship between expression of a major apurinic/apyrimidinic endonuclease (APEX nuclease) and susceptibility to genotoxic agents in human glioma cell lines. 859 68

Saccharomyces cerevisiae Apn1 and Escherichia coli endonuclease IV are homologous enzymes that initiate the repair of abasic (AP) sites or oxidative DNA strand breaks. Yeast lacking Apn1 (apn1-) are hypersensitive to simple alkylating agents (which produce many AP sites) and to oxidants and display an elevated spontaneous mutation rate due to endogenous damages. We explored whether the prokaryotic repair enzyme could substitute for its yeast counterpart. Plasmid constructs were generated that expressed endonuclease IV at 1/20 to 10-fold the AP endonuclease activity of wild-type yeast; some of these plasmids expressed hybrid forms of endonuclease IV equipped with the C-terminal nuclear localization signal of Apn1. Although hybrid endonuclease IV-Apn1 (but not native endonuclease IV) was selectively localized to the yeast nucleus, expression of this chimeric protein at 25% of the normal Apn1 level did not restore alkylation or oxidant resistance to apn1- yeast, but it did partially counteract the mutator phenotype of apn1- yeast. Expression of either the hybrid protein or native endonuclease IV at approximately 10 times the wild-type Apn1 levels restored wild-type resistance to methyl methanesulfonate and near-wild-type H2O2 resistance. High level expression of native endonuclease IV also restored the normal spontaneous mutation rate to apn1- yeast. These data place limits on the amounts of AP endonuclease activity necessary for repair of DNA damages caused by both endogenous and environmental agents and point to a direct role of spontaneous AP sites as potentially mutagenic lesions.
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PMID:Functional expression of Escherichia coli endonuclease IV in apurinic endonuclease-deficient yeast. 863 59

The interaction between mutations at the RAD2 and HIM1 genes was studied. The RAD2 gene encodes endonuclease involved in nucleotide excision repair. Mutants at this gene are highly sensitive to the lethal effect of a variety of mutagens. The product of the HIM1 gene is needed for correction of mismatched bases and repair of premutational DNA damage. Mutations in this gene lead to the formation of the mutator phenotype and high sensitivity to induced mutagenesis. The double rad2 him1 mutant manifested the synergic type of interaction. The level of UV-induced mutagenesis in the double mutant was five times higher than in single mutants, and the absolute yield of forward mutations in five genes controlling adenine biosynthesis was 1 to 2%. UV-induced mutagenesis was increased, at low doses, by several orders of magnitude in the double mutant, compared to the wild-type strain. The high level of mutagenesis in this mutant was caused by ethyl and methyl methanesulfonate. These properties of the stock with the double rad2 him1 mutation makes it promising as a tester in analysis of the gene toxicity of different substances.
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PMID:[A strain of the yeast Saccharomyces cerevisiae for testing environmental mutagens based on interaction of the mutations rad2 and him1]. 872 29

The major genotoxicity of methyl methanesulfonate (MMS) is due to the production of a lethal 3-methyladenine (3MeA) lesion. An alkylation-specific base-excision repair pathway in yeast is initiated by a Mag1 3MeA DNA glycosylase that removes the damaged base, followed by an Apn1 apurinic/ apyrimidinic endonuclease that cleaves the DNA strand at the abasic site for subsequent repair. MMS is also regarded as a radiomimetic agent, since a number of DNA radiation-repair mutants are also sensitive to MMS. To understand how these radiation-repair genes are involved in DNA methylation repair, we performed an epistatic analysis by combining yeast mag1 and apn1 mutations with mutations involved in each of the RAD3, RAD6 and RAD52 groups. We found that cells carrying rad6, rad18, rad50 and rad52 single mutations are far more sensitive to killing by MMS than the mag1 mutant, that double mutants were much more sensitive than either of the corresponding single mutants, and that the effects of the double mutants were either additive or synergistic, suggesting that post-replication and recombination-repair pathways recognize either the same lesions as MAG1 and APN1, or else some differ- ent lesions produced by MMS treatment. Lesions handled by recombination and post replication repair are not simply 3MeA, since over-expression of the MAG1 gene does not offset the loss of these pathways. Based on the above analyses, we discuss possible mechanisms for the repair of methylation damage by various pathways.
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PMID:The repair of DNA methylation damage in Saccharomyces cerevisiae. 893 6

Abasic sites represent ubiquitous DNA lesions that arise spontaneously or are induced by DNA-damaging agents. They block DNA replication and are considered to be cytotoxic and mutagenic. The key enzymes involved in the repair of abasic sites are apurinic/apyrimidinic (AP) endonucleases which process these lesions in an error-free mechanism. To analyze the role of AP endonuclease in the protection of mammalian cells against DNA damaging agents, we have transfected both the human (APE) and the yeast (APN1) AP endonuclease in Chinese hamster cells and compared the effects of expression of these genes in stable transfectants as to survival of cells and formation of chromosomal aberrations. Although APE was markedly expressed on RNA and protein level, nuclear extracts of human APE transfectants did not show a higher AP endonuclease activity than the parental line and became not more resistant to the cell killing and clastogenic effect of methyl methanesulfonate (MMS) and hydrogen peroxide (H2O2). In contrast, cells transfected with the yeast APN1 gene expressed higher AP endonuclease activity and became clearly more resistant to the cytotoxic and chromosome breakage inducing activity of the agents. The results indicate that the excision repair capacity and correspondingly the mutagen resistance can be elevated by introducing, in mammalian cells, a yeast DNA repair gene and verify that AP sites are both cytotoxic and clastogenic lesions.
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PMID:Expression of yeast but not human apurinic/apyrimidinic endonuclease renders Chinese hamster cells more resistant to DNA damaging agents. 908 48

Using complementation tests and nucleotide sequencing, we showed that the rad58-4 mutation was an allele of the MRE11 gene and have renamed the mutation mre11-58. Two amino acid changes from the wild-type sequence were identified; one is located at a conserved site of a phosphodiesterase motif, and the other is a homologous amino acid change at a nonconserved site. Unlike mre11 null mutations, the mre11-58 mutation allowed meiosis-specific double-strand DNA breaks (DSBs) to form at recombination hot spots but failed to process those breaks. DSB ends of this mutant were resistant to lambda exonuclease treatment. These phenotypes are similar to those of rad50S mutants. In contrast to rad50S, however, mre11-58 was highly sensitive to methyl methanesulfonate treatment. DSB end processing induced by HO endonuclease was suppressed in both mre11-58 and the mre11 disruption mutant. We constructed a new mre11 mutant that contains only the phosphodiesterase motif mutation of the Mre11-58 protein and named it mre11-58S. This mutant showed the same phenotypes observed in mre11-58, suggesting that the phosphodiesterase consensus sequence is important for nucleolytic processing of DSB ends during both mitosis and meiosis.
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PMID:A novel mre11 mutation impairs processing of double-strand breaks of DNA during both mitosis and meiosis. 941 73

A dose-limiting toxicity of certain chemotherapeutic alkylating agents is their toxic effects on nontarget tissues such as the bone marrow. To overcome the myelosuppression observed by chemotherapeutic alkylating agents, one approach is to increase the level of DNA repair proteins in hematopoietic stem and progenitor cells. Toward this goal, we have constructed a human fusion protein consisting of O6-methylguanine DNA methyltransferase coupled with an apurinic endonuclease, resulting in a fully functional protein for both O6-methylguanine and apurinic/apyrimidinic (AP) site repair as determined by biochemical analysis. The chimeric protein protected AP endonuclease-deficient Escherichia coli cells against methyl methanesulfonate and hydrogen peroxide (H2O2) damage. A retroviral construct expressing the chimeric protein also protected HeLa cells against 1,3-bis(2-chloroethyl)-1-nitrosourea and methyl methanesulfonate cytotoxicity either when these agents were used separately or in combination. Moreover, as predicted from previous analysis, truncating the amino 150 amino acids of the apurinic endonuclease portion of the O6-methylguanine DNA methyltransferase-apurinic endonuclease protein resulted in the retention of O6-methylguanine DNA methyltransferase activity but loss of all AP endonuclease activity. These results demonstrate that the fusion of O6-methylguanine DNA methyltransferase and apurinic endonuclease proteins into a combined single repair protein can result in a fully functional protein retaining the repair activities of the individual repair proteins. These and other related constructs may be useful for protection of sensitive tissues and, therefore, are candidate constructs to be tested in preclinical models of chemotherapy toxicity.
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PMID:Creation of a fully functional human chimeric DNA repair protein. Combining O6-methylguanine DNA methyltransferase (MGMT) and AP endonuclease (APE/redox effector factor 1 (Ref 1)) DNA repair proteins. 942 28

Abasic (AP) sites arise in DNA through spontaneous base loss and enzymatic removal of damaged bases. APN1 encodes the major AP-endonuclease of Saccharomyces cerevisiae. Human HAP1 (REF1) encodes the major AP endonuclease which, in addition to its role in DNA repair, functions as a redox regulatory protein. We identify APN2, the yeast homolog of HAP1 and provide evidence that Apn1 and Apn2 represent alternate pathways for repairing AP sites. The apn1Delta apn2Delta strain displays a highly elevated level of MMS-induced mutagenesis, which is dependent on the REV3, REV7, and REV1 genes. Our findings indicate that AP sites are highly cytotoxic and mutagenic in eukaryotes, and that the REV3, REV7-encoded DNA polymerase zeta mediates the mutagenic bypass of AP sites.
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PMID:Identification of APN2, the Saccharomyces cerevisiae homolog of the major human AP endonuclease HAP1, and its role in the repair of abasic sites. 976 13

cDNAs for the Xenopus laevis homologue of the endo/exonuclease FEN-1 (DNase IV) have been cloned using a polymerase chain reaction strategy. Products were obtained from two nonallelic Xenopus genes (xFEN-1a and xFEN-1b) that differ from each other by 4.5% in amino acid sequence. Both are 80% identical to mammalian FEN-1 proteins and 55% identical to the yeast homologues. When expressed in Escherichia coli, the Xenopus enzymes showed flap endonuclease activity, a unique feature of this class of nucleases. In addition, expression from the Xenopus cDNAs complemented the temperature and methyl methanesulfonate sensitivity of a yeast rad27 deletion, which eliminates the endogenous FEN-1 gene product. Antiserum raised against xFEN-1 was used to show that the protein accumulates during the middle and late stages of oogenesis, in parallel with other DNA metabolic activities, and that it is localized to the oocyte nucleus. Flap endonuclease activity was demonstrated in oocyte nuclear extracts, and this was inhibited by the anti-xFEN-1 antiserum. The antiserum did not inhibit the major oocyte 5' --> 3' exonuclease activity. DNA synthesis in oocyte extracts was blocked by the antiserum, and the nature of this inhibition suggests that xFEN-1 may be part of a large complex of replication factors. Chromatographic evidence was obtained for the existence of a complex that forms during DNA synthesis and includes proliferating cell nuclear antigen in addition to xFEN-1. These observations support a critical role for xFEN-1 in DNA replication, but indicate that another enzyme must be responsible for the exonuclease function required for homologous recombination in Xenopus oocytes.
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PMID:Characterization of FEN-1 from Xenopus laevis. cDNA cloning and role in DNA metabolism. 985 84


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