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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dose-response curves were compared for deletions [ColBR (resistant to colicin B) mutations being more than 80% deletions] and base changes (reversion of argFam to prototrophy argplus) induced in the same set of E. coli strains (wild-type for DNA repair, uvrA-, polA- and recA-) by N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethyl methanesulfonate (EMS), hydroxylamine (HA), 4-nitroquinoline I-oxide (4NQO), mitomycin C (MTC, UV and X-rays. All these agents induced deletions as well as base changes in the wild-type strain. Thus chemical mutagenesis differed in E. coli and bacteriophages in vitro, for HA, NTG, EMS and perhaps UV produced only point mutations in phage Tr. The patterns of deletion and base-change mutability in E. coli were surprisingly similar. (I) The recombination less recA- strain was mutable by only three (NTG, EMS, HA) of the seven mutagens for either deletions or base changes. (2) The uvrA- strain, unable to excise pyrimidine dimers, was very highly mutable by 4NQO and UV but immutable by MTC for both deletions and base changes. (3) The polA- strain, defective in DNA polymerase I due to a non-suppressible mutation, was very highly mutable by HA and highly mutable by MTC and 4NQO for both deletions and base changes but was highly mutable only for deletions by UV and X-rays, remaining normally mutable by the other agents for both deletions and base changes despite its high sensitivity to their inactivating action. We conclude that errors in the recA-dependent repair of induced DNA damage (after 4NQO, MTC, UV and X-rays) or errors in replication enhanced by damage to the replication system or to the template strands (after NTG, EMS, and HA) give rise to deletions as well as to base changes. From a comparative analysis of 14 dose-response curves for deletions and base changes, we conclude that the order of mutagenic efficiency relative to killing is (EMS, NTG) greater than (UV, 4NQO) greater than HA greater than (X-rays, MTC), and that X-rays, 4NQO, HA and MTC induce more ColBR deletions than Argplus base changes, whereas UV and EMS induce ColBR deletions and Argplus base changes at nearly equal rates and the specificity of NTG is intermediate between these two types.
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PMID:Comparative analysis of deletion and base-change mutabilities of Escherichia coli B strains differing in DNA repair capacity (wild-type, uvrA-, polA-, recA-) by various mutagens. 16 24

Highly selective affinity labeling of the primer site of E. coli DNA polymerase I was performed with the 5'-reactive derivatives of oligothymidylate in the presence of poly(dA) template. Subtilysine cleavage proved that the site of affinity modification belonged to the 'Klenow' part of DNA polymerase I. If taken separately, Klenow fragment was not labeled by these oligonucleotide derivatives. The site of affinity labeling were tested in the structure of DNA polymerase I by hydroxylamine cleavage. At least two sites of labeling were revealed. The main one was localized between Gly-833 and His-928.
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PMID:Highly selective affinity labeling of the primer-binding site of E. coli DNA polymerase I. 222 30

Incubation of the Klenow fragment of E. coli DNA polymerase I with [alpha-32P] dNTP (or NTP) results in the covalent radiolabelling of the enzyme, the bond being stable in acid (pH 2) and alkaline (pH 12) conditions and nucleophiles, such as beta-mercaptoethylamine, efficiently inhibiting the labelling. It is suggested that radiolabelling of the enzyme is the result of formation of chemically active products of the radiolysis of [alpha-32P]NTP (which are likely to be radicals). Non-radioactive NTP hinder the labelling, whereas Mg2+ and polynucleotide do not affect it. Cleavage of the enzyme by hydroxylamine and cyanogen bromide and analysis of gel-electrophoretic patterns of the cleavage products led to conclusion that 32P-label is located between Gly-544 and Met-647.
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PMID:[Covalent labelling of the Klenow fragment of DNA-polymerase I from E. coli]. 269 20

Nitroxide-labeled thymidine substrates (dL) for Escherichia coli DNA polymerase I (pol I) were used to synthesize spin-labeled alternating double-stranded copolymers with (dA-dT)n as a template. All dL substrates use an alkane or alkene tether substituted into the 5-position of the pyrimidine ring to link a five- or six-membered ring nitroxide to the pyrimidine base. The kinetics of dL incorporation show some tether dependence with respect to tether length and tether geometry. The electron spin resonance (ESR) spectra of (dA-dT,dL)n duplexes directly formed by polymerization with pol I are compared with the ESR spectra of (dA)n(dT,dL)n duplexes, which are obtained after annealing of nitroxide-labeled single strands with complementary unlabeled single strands. The ESR spectra indicate that nitroxide-labeled analogues with tethers short enough to let the nitroxide ring reside in the major groove are excellent reporter groups for monitoring hybridization. A small difference between the ESR line shapes of the alternating duplexes (dA-dT,dL)n and the homopolymer duplexes (dA)n(dT,dL)n containing the same dL is detectable, suggesting the presence of subtle differences in the base dynamics between both systems. Computer simulation of the ESR spectra of the (dA-dT,dL)n duplexes was successful with the same motional model reported earlier [Kao, S.-C., & Bobst, A.M. (1985) Biochemistry 24, 5465-5469]. The thymidine motion arising from tilting and torsion of base pairs and base twisting in (dA-dT)n is similar to that in (dA)n(dT)n and is of the order of 4 ns.
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PMID:Base dynamics of nitroxide-labeled thymidine analogues incorporated into (dA-dT)n by DNA polymerase I from Escherichia coli. 282 51

Aphidicolin-resistant mutants (Aphr) of Bacillus subtilis bacteriophage phi 29 were isolated after mutagenesis with hydroxylamine. Efficiency of plating (e.o.p.) of the resistant mutants was not reduced at 500 microM aphidicolin, although e.o.p. of wild type phi 29 was less than 10(-5) at the same concentration of aphidicolin. By recombination and complementation analyses, both sites of the mutations, aph-71 and aph-101, of Aphr71 and Aphr101, respectively, were mapped in gene 2 which encodes phi 29 DNA polymerase. The activity of wild type phi 29 DNA polymerase, in a partially purified fraction, was inhibited by aphidicolin. DNA polymerases from Aphr71 and Aphr101, prepared in the same manner as that of wild type, were resistant to the drug. These results indicate that the acquisition of the aphidicolin resistance of Aphr71 and Aphr101 of bacteriophage phi 29 results from a structural alteration of phi 29 DNA polymerase which reduces sensitivity to aphidicolin.
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PMID:Aphidicolin-resistant mutants of bacteriophage phi 29: genetic evidence for altered DNA polymerase. 308 58

Two antimutagenic DNA polymerases of bacteriophage T4 markedly reduce transition mutagenesis by a variety of chemical mutagens. Spontaneous mutation and mutagenesis by 2-aminopurine, 5-bromodeoxyuridine, and thymine deprivation are strongly suppressed. Mutagenesis at G:C sites by ethyl methanesulfonate, and at A:T sites by nitrous acid, is moderately suppressed. Mutagenesis at G:C sites by hydroxylamine and by nitrous acid is not suppressed. These results support the notion that the indispensable DNA polymerase of bacteriophage T4 plays a crucial role in the selection of the correct base during DNA replication. The data also reveal that mutagenic specificities of chemical agents depend as much upon the characteristics of the enzymatic apparatus of DNA replication as they do upon the chemistry of primary mutational lesions.
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PMID:Suppression of chemical mutagenesis in bacteriophage T4 by genetically modified DNA polymerases. 526 45

We report on the properties of a temperature-sensitive mutant produced by transfection of cells with intact DNA and a specific DNA fragment mutagenized with low levels of hydroxylamine. The plating efficiency of the mutant at 39 degrees C relative to that at 33.5 degrees C was 5 X 10(-6). The pattern of polypeptides produced at the nonpermissive temperature was similar to that seen with wild-type virus in infected cells treated with inhibitory concentrations of phosphonoacetic acid in that alpha and beta polypeptides were produced, whereas most gamma polypeptides were either reduced or absent. Consistently, the mutant did not make viral DNA, although temperature sensitivity of the viral DNA polymerase could not be demonstrated. Marker rescue studies with herpes simplex virus type 2 (HSV-2) DNA mapped the mutant in the L component within map positions 0.385 and 0.402 in the prototype (P) arrangement of the HSV-1 genome. Analysis of the recombinants permitted the mapping of the genes specifying infected cell polypeptides 36, 35, 37, 19.5, 11, 8, 2, 43, and 44, but only the infected cell polypeptide 8 of HSV-2 was consistently made by all recombinants containing demonstrable HSV-2 sequences. Marker rescue studies with cloned HSV-1 DNA fragments mapped the temperature-sensitive lesion within less than 10(3) base pairs between 0.383 and 0.388 map units. Translation of the RNA hybridizing to cloned HSV-1 DNA, encompassing the smallest region containing the mutation, revealed polypeptide 8 (128,000 molecular weight), which was previously identified as a beta polypeptide with high affinity for viral DNA, and a polypeptide (25,000 molecular weight) not previously identified in lysates of labeled cells.
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PMID:Molecular genetics of herpes simplex virus. VII. Characterization of a temperature-sensitive mutant produced by in vitro mutagenesis and defective in DNA synthesis and accumulation of gamma polypeptides. 626 Sep 73

The most direct approach to elucidating the roles of herpes simplex virus (HSV) proteins in the viral replicative cycle has been to isolate temperature-sensitive, cytolysis-resistant, and drug-resistant mutants that exhibit alterations in the synthesis or activity of these proteins. The development of procedures for the introduction of temperature-sensitive mutations into physically defined regions of the viral genome and for fine mapping of these mutations has proven especially valuable. Thus, (1) hydroxylamine mutagenesis of the HSV-1 BglII I fragment (coordinates 0.312-0.415) has facilitated the genetic and functional characterization of the gene for the major viral DNA-binding protein of 130 K molecular weight; (2) the selection of a mutant conditionally able to render infected cells resistant to immune cytolysis has led to identification of an HSV gene involved in the processing of viral glycoproteins; and (3) the combined use of temperature-sensitive and drug-resistant mutants has led to a better definition of the physical limits and functional domains of the gene for HSV DNA polymerase.
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PMID:Genetics of herpes simplex virus. 633 Feb 20

It has been reported that single stranded viral DNA reacts with the carcinogen, chloroacetaldehyde at specific hot spots (Premaratne et al., 1993 Int. J. Biochem. 25, 1669-1672). We tested this occurrence with several other mutagens and potential carcinogens. A series of chemicals (chloroacetaldehyde, methyl, ethyl, and propyl nitro nitrosoguanidine, hydrazine, 2,4 dinitrophenyl hydrazine, hydroxylamine and methyl methanesulfonate) were each separately reacted with viral M13mp18 DNA for 2 hr at 37 degrees C and pH 4.9. The locations of adduction were identified as points of chain termination (or polymerase fall off) when the reacted DNA was subjected to a modified sequencing procedure that had ample regular labeled and unlabeled nucleotides but lacked dideoxy chain termination mixtures. Chain termination was observed to occur at specific, non-random, sites rather than with equal probability at all bases of the DNA. Chemicals with similar structures had identical points of "fall off". The pattern of chain termination appears to be unique to each class of compounds and is independent of temperature, pH, and salt concentration. Termination is believed to occur when the DNA polymerase encounters an adduct. Mutagens of different unrelated structures when reacted with this DNA produced different sites of adduct formation, while the alkyl nitro nitrosoguanidines, compounds with homologous structure showed identical points of chain termination.
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PMID:Detection of mutagen specific adduct formation in DNA using sequencing methodology. 758 13

Protein splicing is a self-catalyzed, posttranslational process which converts a precursor polypeptide into two new proteins by the excision of an internal polypeptide segment and the ligation of the flanking polypeptides. Evidence has been presented that protein splicing involves a branched intermediate, which is resolved into the two protein products by the cyclization of an asparagine residue to aminosuccinimide [Xu, M. Q., Comb, D. G., Paulus, H., Noren, C. J., Shao, Y., & Perler, F. (1994) EMBO J. 13, 5517-5522]. This report describes the chemical synthesis of a peptide with a C-terminal aminosuccinimide residue, corresponding to the putative C-terminus of the excised intervening sequence (intein) derived from the thermostable DNA polymerase of Pyrococcus species GB-D. The synthetic aminosuccinimide peptide was compared with the C-terminal cyanogen bromide peptide of the excised intein and found to be indistinguishable in terms of its chromatographic properties, high-resolution mass spectrum, and colorimetric assay involving reaction with hydroxylamine. This establishes definitively that protein splicing is accompanied by the cyclization of asparagine to yield an aminosuccinimide residue at the C-terminus of the excised intein and that this unusual residue is therefore a natural constituent of spliced proteins. The effects of pH and temperature on the stability of the synthetic aminosuccinimide peptide are described.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Protein splicing: characterization of the aminosuccinimide residue at the carboxyl terminus of the excised intervening sequence. 766 64


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