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)

Ether-permeabilized (nucleotide-permeable) Escherichia coli cells respond to alkylating and arylalkylating carcinogens with DNA excision repair, as assessed by their stimulation of DNA repair synthesis. In the present work, we have investigated whether DNA repair synthesis in ether-treated E. coli cells can serve as a general indicator to monitor the DNA-binding of carcinogens, mutagens and antitumor agents. Therefore, a standard assay was developed and comparative analyses were performed on 11 ultimate carcinogens, 10 proximate carcinogens, 2 tumor promoters, 6 mutagens, and 12 antitumor agents. All ultimate carcinogens (alkylating, acylating, arylalkylating agents) and mutagens (e.g., hydrogeen peroxide, acridine derivatives) caused DNA excision repair in wild type cells as measured by [3H] dTMP incorporation and simultaneously inhibited replicative DNA synthesis to various extents. Control experiments with the mutant cells uvrA and uvrB were performed to determine whether the pyrimidine-dimer-specific UV-endonuclease was involved in the removal of DNA damage. This was found to be true for the ultimate carcinogens (Ac)2 ONFln, mitomycin C, and for very reactive alkylating carcinogens. None of the ultimate carcinogens induced repair polymerization in mutant cells lacking the 5'-3' exonucleolytic activity of DNA polymerase I. Proximate carcinogens, such as Me2NNO, 4-nitroquinoline-1-oxide and aflatoxins, did not induce excision repair in the standard assay, probably because of the inability of E. coli to perform the activation steps necessary for covalent DNA-binding. However, Me2NNO, when pretreated with Udenfriend's hydroxylating mixture, gave rise to a low level of repair polymerization in ether-treated cells. Intercalating mutagens, such as quinacrine and ethidum bromide, inhibited replicative DNA synthesis. However, they were not found to be repair-inducers. THE TUMOR PROMOters TPA and phorbol-12,13-didecanoate did not cause excision repair, even when applied at high concentrations, nor did they inhibit repair synthesis stimulated by MeNOUr or (Ac)2 ONFln. The antitumor agents may be classified into two groups on the basis of the influence they exert on DNA synthesis: members of the first group (involving BCNU and bleomycin) stimulate repair polymerization and, in addition, inhibit DNA replication. These compounds are known to bind covalently to DNA. The second group of drugs (including adriamycin and cis-Pt(II)diammine complexes) inhibits DNA replication without stimulating repair synthesis. The predominant DNA-interaction of these compounds is known to be a non-covalent (i.e., intercalative, electrostatic) binding. Our experiments show that the ether-permeabilized E. coli cell can be successfully used to test ultimate carcinogens, mutagens and antitumor agents for repair-inducing and replication-inhibiting activity. The standard test might be extended to pre- and proximate carcinogens, provided these can be suitably activated.
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PMID:The nucleotide-permeable Escherichia coli cell, a sensitive DNA repair indicator for carcinogens, mutagens, and antitumor agents binding covalently to DNA. 15 98

DNA polymerase-alpha and -beta can be distinguished from one another by the differential effects of N-ethylmaleimide, KCl, ara-CTP and temperature, as well as on the basis of sedimentation. The sensitivity of DNA polymerase-beta to elevated temperatures as compared to DNA polymerase-alpha provides a new means of distinguishing between these two enzymes even in crude extracts and a possible probe for determining their function. DNA polymerase-alpha and -beta share several properties in common, including the ability to readily incorporate dUTP in place of dTTP. The Km for dUTP varies from 10 to 30 micron with different preparations of DNA polymerase-alpha and -beta. Thus, in mammalian cells, dUMP could be incorporated into DNA, and if excised by an endonuclease, would lead to discontinuities. Initial analyses of fidelity in direct comparative studies indicate that beta-class DNA polymerases are highly accurate in base selection when copying poly[d(A-T)]. Less than one molecule of dGMP is incorporated for every 12 000-45 000 molecules of dAMP and dTMP polymerized. DNA polymerase-alpha is somewhat less accurate, making one mistake for every 4000-10 000 correct nucleotides incorporated. Since both polymerases lack an exonucleolytic activity, this accuracy must be the result of selectivity for the complementary nucleotide by the polymerase.
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PMID:Distinctive properties of mammalian DNA polymerases. 28 7

In 1983, a small outbreak of infections caused by a previously unrecognized multiply-drug-resistant Shigella flexneri 3a strain occurred on the Hopi Indian reservation. The index patient, a diabetic woman with recurrent Escherichia coli bacteriuria on prophylactic trimethoprim/sulfamethoxazole (TMP/SMZ) therapy, was hospitalized with concurrent E. coli urinary tract infection and shigellosis. Both E. coli isolated from her urine and S. flexneri isolated from her stool were resistant to ampicillin, carbenicillin, streptomycin, sulfisoxazole, tetracycline, and TMP/SMZ. Both isolates contained a 35-MDa plasmid transferrable to recipient E. coli, and transconjugates acquiring the plasmid from either donor strain also acquired resistance to the same agents. The number and size of fragments generated by plasmid digestion with DNA restriction endonuclease ClaI were similar. A review of clinical microbiology records showed that an E. coli strain isolated from the patient 3 w before the onset of shigellosis had identical antimicrobial resistance to the E. coli and Shigella isolated during the outbreak. These studies indicate that the index patient receiving prophylactic TMP/SMZ was the likely source of the R-plasmid for the outbreak strain of Shigella.
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PMID:Interspecies gene transfer in vivo producing an outbreak of multiply resistant shigellosis. 268 26

The percentage of clinical isolates of several species of Enterobacteriaceae, particularly Escherichia coli and Klebsiella pneumoniae, resistant to trimethoprim (TMPR) has increased gradually at the Brigham and Women's Hospital (Boston) in recent years. Thirty-seven of 42 TMPR isolates from six species of gram-negative bacilli conjugally transferred TMP resistance to K12 E. coli. beta-Lactam resistance cotransferred from 21 of the 37 donors, and sulfamethoxazole (SMZ) resistance cotransferred from five of the 37 donors. Plasmids that encoded TMP resistance either alone or with SMZ resistance had a molecular size of approximately 52.5 kilobases, with identical restriction endonuclease-generated "fingerprints." Plasmids encoding beta-lactam-mediated resistance (beta R) were approximately four kilobases larger and had fragment patterns that were identical for all of the TMPR/beta R plasmids tested and had many restriction endonuclease-generated bands in common with TMPR plasmids. Radiolabeled dihydrofolate reductase (DHFR) probes identified the type II DHFR as the determinant of TMP resistance. In contrast with reports from Europe, TMP resistance in multiple species of Enterobacteriaceae was found to be spread in one hospital by a single, stable conjugative plasmid that has a wide host range and encodes the type II DHFR gene.
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PMID:Trimethoprim resistance in multiple genera of Enterobacteriaceae at a U.S. hospital: spread of the type II dihydrofolate reductase gene by a single plasmid. 298 9

Linear phiX174 single-stranded DNA can be isolated from phiX phage particles produced under various conditions. About half of the linear strands have a dGMP residue at the 5' end, the remaining have roughly comparable amounts of dCMP, dTMP, and dAMP. The linear strands can be converted to covalently closed circular molecules by polynucleotide ligase, but only after they have been incubated with T4 DNA polymerase and deoxynucleoside triphosphates. Experiments with endonuclease R, the restriction enzyme from Haemophilus influenzae, indicated that the nucleotides incorporated into the DNA during this reaction were found predominantly in a limited region of the genome. The results suggest that the normal intermediate in single-stranded phiX174 DNA synthesis may be a single-stranded linear molecule which is shorter than unit length and is intrinsically capable of circularization.
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PMID:Mechanism of replication of single-stranded PhiX174 DNA. VII. Circularization of the progeny viral strand. 459 Oct 49

Heat treatment of poly(deoxycytidylic acid)-[poly(dC)] induces the formation of dUMP residues, which code for dAMP when replicated by Escherichia coli DNA polymerases I and III. The specificity of dUMP coding properties is indicated by the quantitative relation between the dAMP incorporated and the frequency of dUMP residues in the heat-treated poly(dC). The dAMP incorporation is prevented by preincubation of uracil containing poly(dC) with uracil-DNA glycosylase. The excision of uracil by uracil-DNA glycosylase leads to the formation of apyrimidinic sites (AP sites), which are barely replicated in vitro under physiological conditions. However, the alteration of E. coli DNA polymerase I fidelity of replication by Mn2+ greatly stimulates the replication of AP sites. There is a preferential incorporation of dAMP, as compared to dTMP, opposite the AP sites. The dAMP incorporation is prevented by preincubation of poly(dC) containing AP sites with Micrococcus luteus AP endonuclease B. The results show a close association between DNA repair by base excision and the prevention of mutagenic processes in vitro. Furthermore, since the alteration of DNA polymerase fidelity allows some replication of the noncoding DNA lesion (AP site), this could imply a role in SOS-induced mutagenesis in vivo.
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PMID:Coding properties of poly(deoxycytidylic acid) templates containing uracil or apyrimidinic sites: in vitro modulation of mutagenesis by deoxyribonucleic acid repair enzymes. 676 Aug 93

Transfer of shigella R-plasmids in vivo has seldom been demonstrated. Strains of Shigella dysenteriae type 1 and Shigella flexneri type 5b were isolated from a Bulgarian traveller who visited Vietnam and developed dysentery, which was treated with trimethoprim/sulfamethoxazole (TMP/SMZ) for a short time. Both species of shigellae are unusual in Bulgaria where strains of S. sonnei predominate. Both shigella strains were multiresistant to the same antimicrobial agents. Each strain contained a 48-kilobase plasmid that conferred the entire resistance phenotype to a susceptible Escherichia coli. Restriction endonuclease patterns of plasmid DNA from the respective strains were identical. Transmissible plasmids of the same resistance phenotypes and restriction patterns were isolated from the patient's colonic E. coli. Transconjugants hybridized to a dihydrofolate reductase type I-DNA probe. These studies support the hypothesis that R-plasmid transfer may occur between non-pathogenic, faecal strains and pathogenic shigellae, a process that may have been facilitated by inadequate treatment with TMP/SMZ at the onset of the illness.
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PMID:In vivo R-plasmid transfer in a patient with a mixed infection of shigella dysentery. 814 99

DNA polymerases (deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase EC 2.7.7.7.) were extracted from regenerating livers from young and aged rats. DNA polymerase alpha was separated and partially purified by DEAE-cellulose column chromatography, polyethyleneglycol precipitation, and phosphocellulose column chromatography, and fidelity levels were then monitored with the synthetic template-primer poly (dG-dC). The fidelity level of the DNA polymerase from regenerating liver a 4-month-old rat was very high, while that of the DNA polymerase from a 24-month-old rat was significantly decreased. To confirm this result, DNA was synthesized on poly (dG-dC) in a reaction mixture containing [32P]dTTP, and the synthetic polynucleotide was purified and digested with HhaI restriction endonuclease. After hydrolysis, the oligonucleotides were developed by two dimensional thin layer chromatography on PEI cellulose plates. Spots containing [32P]dTMP were observed when DNA polymerase from a 24 month-old rat was used, but none was found in polynucleotides synthesized using DNA polymerase from a 4 month-old rat. Nearest neighbor analysis suggested that dG-dT and dC-dT pairs were constructed by mis-incorporation due to DNA polymerase alpha.
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PMID:Age-associated changes in the template-reading fidelity of DNA polymerase alpha from regenerating rat liver. 908 Mar 95

Abasic [apurinic/apyrimidinic (AP)] sites are common, noncoding DNA lesions. Despite extensive investigation, the mutational pattern they provoke in eukaryotic cells remains unresolved. We constructed Saccharomyces cerevisiae strains in which chromosomal AP sites were generated during normal cell growth by altered human uracil-DNA glycosylases that remove undamaged cytosines or thymines. The mutation target was the URA3 gene inserted near the ARS309 origin to allow defined replication polarity. Expression of the altered glycosylases caused a 7- to 18-fold mutator effect in AP endonuclease-deficient (deltaapn1) yeast, which depended highly on the known translesion synthesis enzymes Rev1 and DNA polymerase zeta. For the C-glycosylase, GC>CG transversions were the predominant mutations, followed by GC>AT transitions. AT>CG transversions predominated for the T-glycosylase. These results support a major role for Rev1-dependent dCMP insertion across from AP sites and a lesser role for dAMP insertion. Unexpectedly, there was also a significant proportion of dTMP insertions that suggest another mutational pathway at AP sites. Although replication polarity did not strongly influence mutagenesis at AP sites, for certain mutation types, there was a surprisingly strong difference between the transcribed and non-transcribed strands of URA3. The basis for this strand discrimination requires further exploration.
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PMID:Mutagenic specificity of endogenously generated abasic sites in Saccharomyces cerevisiae chromosomal DNA. 1631 79

Human DNA apurinic/apyrimidinic (AP-) endonuclease 1 (APE1) is involved in the base excision repair (BER) pathway. The enzyme hydrolyzes DNA from the 5 side of the AP site. In addition to endonuclease activity, APE1 also possesses other slight activities including 3 -5 exonuclease activity. The latter is preferentially exhibited towards mispaired (non-canonical) nucleotides, this being the reason why APE1 is considered as a proofreading enzyme correcting the misincorporations introduced by DNA polymerase beta. We have studied 3 -5 exonuclease activity of APE1 towards dCMP and dTMP residues and modified dCMP analogs with photoreactive groups at the 3 end of the nicked DNA. Photoreactive dNMP residues were incorporated at the 3 end of the lesion using DNA polymerase beta and photoreactive dNTPs. The dependence of exonuclease activity on the "canonicity" of the base pair formed by dNMP flanking the nick at the 3 end, on the nature of the group flanking the nick at the 5 end, and on the reaction conditions has been determined. Optimal reaction conditions for the 3 -5 exonuclease hydrolysis reaction catalyzed by APE1 in vitro have been established, and conditions when photoreactive residues are not removed by APE1 have been chosen. These reaction conditions are suitable for using photoreactive nicked DNAs bearing 3 -photoreactive dNMP residues for photoaffinity labeling of proteins in cellular/nuclear extracts and model APE1-containing systems. We recommend using FAPdCTP for photoaffinity modification in APE1-containing systems because the FAPdCMP residue is less prone to exonuclease degradation, in contrast to FABOdCTP, which is not recommended.
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PMID:3'-5' exonuclease activity of human apurinic/apyrimidinic endonuclease 1 towards DNAs containing dNMP and their modified analogs at the 3 end of single strand DNA break. 1648 26


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