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)

More than twenty repeating sequence DNAs containing phosphorothioates were prepared from the appropriate dXTPs with DNA polymerase I. The Tms of the modified DNAs were all lower than the parent polymers. A phosphorothioate group 5' to a pyrimidine gave rise to a large decrease than 5' to a purine, e.g., poly(dA).poly(dT) = 50 degrees; poly(dsA).poly(dT) = 44 degrees; poly(dA).poly(dsT) = 33 degrees; and poly(dsA).poly(dsT) = 26 degrees. The presence of phosphorothioate groups had a dramatic effect on triplex formation; poly[d(TC)].poly[d(sGsA)] spontaneously dismutases to a triplex at pH 8 whereas triplex formation in poly[d(sTsC)].poly[d(GA)] was inhibited. Surprisingly poly(dsG).poly(dC) had a Tm which initially decreased with increasing ionic strength. Resistance to digestion with pancreatic DNAse I did not correlate with phosphorothioate content. Poly[d(AsT)], poly[d(TsC)].poly[d(sGA)] and poly[d(sTG)].poly[d(sCA)] were resistant whereas poly[d(sAT)] and poly[d(sTsTG)].poly[d(CsAsA)] were rapidly degraded. Thus phosphorothioate groups cause small conformational changes and may reveal new families of conformational polymorphisms.
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PMID:Synthetic repeating sequence DNAs containing phosphorothioates: nuclease sensitivity and triplex formation. 292 86

Replication of UV-irradiated oligodeoxynucleotide-primed single-stranded phi X174 DNA with Escherichia coli DNA polymerase III holoenzyme in the presence of single-stranded DNA-binding protein was investigated. The extent of initiation of replication on the primed single-stranded DNA was not altered by the presence of UV-induced lesions in the DNA. The elongation step exhibited similar kinetics when either unirradiated or UV-irradiated templates were used. Inhibition of the 3'----5' proofreading exonucleolytic activity of the polymerase by dGMP or by a mutD mutation did not increase bypass of pyrimidine photodimers, and neither did purified RecA protein influence the extent of photodimer bypass as judged by the fraction of full length DNA synthesized. Single-stranded DNA-binding protein stimulated bypass since in its absence the fraction of full length DNA decreased 5-fold. Termination of replication at putative pyrimidine dimers involved dissociation of the polymerase from the DNA, which could then reinitiate replication at other available primer templates. Based on these observations a model for SOS-induced UV mutagenesis is proposed.
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PMID:Mechanism of replication of ultraviolet-irradiated single-stranded DNA by DNA polymerase III holoenzyme of Escherichia coli. Implications for SOS mutagenesis. 294 23

Replication of UV-irradiated circular single-stranded phage M13 DNA by Escherichia coli RNA polymerase (EC 2.7.7.6) and DNA polymerase III holoenzyme (EC 2.7.7.7) in the presence of single-stranded DNA binding protein yielded full-length as well as partially replicated products. A similar result was obtained with phage G4 DNA primed with E. coli DNA primase, and phage phi X174 DNA primed with a synthetic oligonucleotide. The fraction of full-length DNA was several orders of magnitude higher than predicted if pyrimidine photodimers were to constitute absolute blocks to DNA replication. Recent models have suggested that pyrimidine photodimers are absolute blocks to DNA replication and that SOS-induced proteins are required to allow their bypass. Our results demonstrate that, under in vitro replication conditions, E. coli DNA polymerase III holoenzyme can insert nucleotides opposite pyrimidine dimers to a significant extent, even in the absence of SOS-induced proteins.
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PMID:Replication of UV-irradiated single-stranded DNA by DNA polymerase III holoenzyme of Escherichia coli: evidence for bypass of pyrimidine photodimers. 294 56

We have examined the induction of alkali-labile regions in DNA of human neoplastic cells treated with 5-fluorouracil and 5-fluorodeoxyuridine. 5-Fluorouracil induces DNA lesions by two mechanisms: incorporation of drug into DNA and a second mechanism not involving the incorporation. The second mechanism is seen in cells treated with aphidicolin, a specific inhibitor of DNA polymerase alpha, to stop the movement of the DNA replication forks. 5-Fluorodeoxyuridine is not incorporated into DNA of these cells; only the second mechanism of induction of alkali-labile DNA is detected. The second mechanism is in all probability due to inefficient DNA repair of normally occurring defects in purine and pyrimidine residues. Furthermore there is a correlation between increasing levels of alkali-labile regions in the DNA and cytotoxicity of the drugs. This may be one explanation for the cytocidal effects of 5-fluoropyrimidines.
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PMID:DNA lesions in human neoplastic cells and cytotoxicity of 5-fluoropyrimidines. 294 36

During in vitro replication of UV-irradiated single-stranded DNA with Escherichia coli DNA polymerase III holoenzyme termination frequently occurs at pyrimidine photodimers. The termination stage is dynamic and characterized by at least three different events: repeated dissociation-reinitiation cycles of the polymerase at the blocked termini; extensive hydrolysis of ATP to ADP and inorganic phosphate; turnover of dNTPs into dNMP. The reinitiation events are nonproductive and are not followed by further elongation. The turnover of dNTPs into dNMPs is likely to result from repeated cycles of insertion of dNMP residues opposite the blocking lesions followed by their excision by the 3'----5' exonucleolytic activity of the polymerase. Although all dNTPs are turned over, there is a preference for dATP, indicating that DNA polymerase III holoenzyme has a preference for inserting a dAMP residue opposite blocking pyrimidine photodimers. We suggest that the inability of the polymerase to bypass photodimers during termination is due to the formation of defective initiation-like complexes with reduced stability at the blocked termini.
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PMID:Dynamics of termination during in vitro replication of ultraviolet-irradiated DNA with DNA polymerase III holoenzyme of Escherichia coli. 295 58

The RecA protein of Escherichia coli is required for SOS-induced mutagenesis in addition to its recombinational and regulatory roles. We have suggested that RecA might participate directly in targeted mutagenesis by binding preferentially to the site of the DNA damage (e.g. pyrimidine dimer) because of its partially unwound nature; DNA polymerase III will then encounter RecA-coated DNA at the lesion and might replicate across the damaged site more often but with reduced fidelity. In support of this proposal, we have found that the phenotype of wild-type and mutant RecA for mutagenesis correlates with capacity to bind to double-stranded DNA. Wild-type RecA binds more efficiently to ultraviolet (u.v.)-irradiated, duplex DNA than to non-irradiated DNA. The RecA441 (Tif) protein that is constitutive for mutagenesis binds extremely well to double-stranded DNA with no lesions, whereas the RecA430 protein that is defective in mutagenesis binds poorly even to u.v.-irradiated DNA. The RecA phenotype also correlates with capacity to use duplex DNA as a cofactor for cleavage of the LexA repressor protein for SOS-controlled operons. Wild-type RecA provides efficient cleavage of LexA only with u.v.-irradiated duplex DNA; RecA441 cleaves well with non-irradiated DNA; RecA430 gives very poor cleavage even with u.v.-irradiated DNA. We conclude that the interaction of RecA with damaged double-stranded DNA is likely to be a critical component of SOS mutagenesis and to define a pathway for the LexA cleavage reaction as well.
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PMID:RecA protein and SOS. Correlation of mutagenesis phenotype with binding of mutant RecA proteins to duplex DNA and LexA cleavage. 296 Aug 17

pBR322 DNA, linearized by lysis of an oxolinic acid-treated culture of Escherichia coli strain DK6recA- (pBR322) with sodium dodecyl sulfate, was purified, treated with DNA polymerase in the presence of the four deoxynucleoside triphosphates, and ligated to DNA linkers containing the XhoI recognition sequence. Most of the drug-resistant colonies resulting from transformation of E. coli with this material bore plasmids that appeared by restriction enzyme analysis to differ from pBR322 only by the introduction of an XhoI site. The XhoI sites in plasmids from 93 transformants were distributed unevenly around the pBR322 map. Maxam-Gilbert DNA sequence analysis of 36 of these plasmids, labeled at the 5' termini of the XhoI sites, revealed that 29 of them contained, in addition to the XhoI linker, a duplication of four base-pairs of the pBR322 sequence surrounding the linker. Therefore, oxolinic acid-induced linearization must have resulted in 5'-terminal extensions of four bases, the configuration known to result from oxolinic acid-induced DNA cleavage by DNA gyrase in vitro. The sequence data thus allowed the determination of the precise point at which linearization occurred, apparently by the abortion of a gyrase-DNA covalent intermediate that existed in vivo. When the 19 different sites of the 29 plasmids were compared, the following set of rules could be derived: (formula; see text) where N is any nucleotide, R is a purine, and Y is a pyrimidine. Cleavage occurred at the line between the eighth and ninth positions from the left. The parenthetical G and T were preferred secondarily to T and G, respectively, whereas T and G in the 13th position from the left were equally preferred. Several of these rules are similar to those proposed previously based on several in vitro gyrase cleavage sites. Some of our rules show dyad symmetry around the axis midway between the cleavage points in the two strands, while others are distinctly asymmetric.
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PMID:Sites of reaction of Escherichia coli DNA gyrase on pBR322 in vivo as revealed by oxolinic acid-induced plasmid linearization. 298 30

Ultraviolet light irradiation of DNA results in the formation of two major types of photoproducts, cyclobutane dimers and 6-4' [pyrimidin-2'-one] -pyrimidine photoproducts. The enzyme T4 DNA polymerase possesses a 3' to 5' exonuclease activity and hydrolyzes both single and double stranded DNA in the absence of deoxynucleotide triphosphate substrates. Here we describe the use of T4 DNA polymerase associated exonuclease for the detection and quantitation of UV light-induced damage on both single and double stranded DNA. Hydrolysis of UV-irradiated single or double stranded DNA by the DNA polymerase associated exonuclease is quantitatively blocked by both cyclobutane dimers and (6-4) photoproducts. The enzyme terminates digestion of UV-irradiated DNA at the 3' pyrimidine of both cyclobutane dimers and (6-4) photoproducts. For a given photoproduct site, the induction of cyclobutane dimers was the same for both single and double stranded DNA. A similar relationship was also found for the induction of (6-4) photoproducts. These results suggest that the T4 DNA polymerase proofreading activity alone cannot remove these UV photoproducts present on DNA templates, but instead must function together with enzymes such as the T4 pyrimidine dimer-specific endonuclease in the repair of DNA photoproducts. The T4 DNA polymerase associated exonuclease should be useful for the analysis of a wide variety of bulky, stable DNA adducts.
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PMID:T4 DNA polymerase (3'-5') exonuclease, an enzyme for the detection and quantitation of stable DNA lesions: the ultraviolet light example. 298 81

DNA damage and repair in human cells exposed to ultraviolet light (254 nm) or to psoralen derivatives plus 360 nm light were compared by means of a variety of analytic techniques. The two kinds of damage show considerable structural similarity; both involve cyclobutyl bonds to 5,6 positions of pyrimidines as major products and have various minor products. In purified DNA, pyrimidine dimers, but not psoralen adducts, cause structural distortions that are substances for digestion with single-strand-specific nucleases. Whereas pyrimidine dimers are randomly produced in chromatin, psoralen adducts, are concentrated approximately 2- to 4-fold in linker regions of chromatin at doses that are not highly lethal. Chromatin shows considerable mobility; assignment of DNA to linker or core regions is not permanent, and psoralen adducts initially concentrated in linker regions become randomized after 10 hr. Pyrimidine dimers and psoralen adducts are excised by normal cells but not by repair-deficient xeroderma pigmentosum cells. This repair process requires DNA polymerase alpha, but its rate in ultraviolet-damaged cells is twice that in psoralen-damaged cells. Conversion of monoadducts to DNA-DNA crosslinks reduces the rate of repair because of the increased complexity of the damaged site.
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PMID:Formation and repair of psoralen-DNA adducts and pyrimidine dimers in human DNA and chromatin. 300 74

Purine ribonucleoside monophosphates were found to inhibit chain elongation catalyzed by herpes simplex virus (HSV) DNA polymerase when DNA template-primer concentrations were rate-limiting. Inhibition was fully competitive with DNA template-primer during chain elongation; however, DNA polymerase-associated exonuclease activity was inhibited noncompetitively with respect to DNA. Combinations of 5'-GMP and phosphonoformate were kinetically mutually exclusive in dual inhibitor studies. Pyrimidine nucleoside monophosphates and deoxynucleoside monophosphates were less inhibitory than purine riboside monophosphates. The monophosphates of 9-beta-D-arabinofuranosyladenine, Virazole (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), 9-(2-hydroxyethoxymethyl)guanine, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine exerted little or no inhibition. In contrast to HSV DNA polymerase, human DNA polymerase alpha was not inhibited by purine ribonucleoside monophosphates. These studies suggest the possibility of a physiological role of purine ribonucleoside monophosphates as regulators of herpesvirus DNA synthesis and a new approach to developing selective anti-herpesvirus compounds.
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PMID:Inhibition of herpes simplex virus DNA polymerase by purine ribonucleoside monophosphates. 300 73


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