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)

A human fibroblast nick translation assay has been applied to an examination of 48 diverse chemical agents to assess their ability to specifically interfere with the DNA excision-repair process following ultraviolet irradiation. Certain inhibitors of DNA polymerase, ribonucleotide reductase and purine and pyrimidine biosynthesis are shown to inhibit the resynthesis step of repair while DNA intercalators and inhibitors of DNA topoisomerases appear to inhibit the incision step. A variety of other agents previously implicated as inhibitors of DNA repair was also examined and found to have no such effect. This type of analysis should prove useful in the rapid identification of new classes of compounds that antagonize normal cellular repair functions and that might, therefore, act as comutagens or cocarcinogens.
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PMID:Evaluation of putative inhibitors of DNA excision repair in cultured human cells by the rapid nick translation assay. 300 55

Replication of equine herpesvirus type 1 (EHV-1) was sensitive to 9-(1,3-dihydroxy-2-propoxymethyl)guanine(DHPG) but relatively resistant to E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU). Likewise, plaque formation by EHV-1 was inhibited by DHPG, but not by BVDU. Plaque formation by a thymidine kinase-negative (tk-) mutant of EHV-1 was not inhibited by DHPG. In order to investigate biochemical mechanisms determining the differential sensitivity of EHV-1 to these drugs, the EHV-1-encoded thymidine kinase enzyme activity (TK)1 was partially purified from EHV-1-infected cells and analyzed. The EHV-1-induced enzyme utilized both ATP and CTP as phosphate donors and differed in relative electrophoretic mobility from the TKs of mock-infected and HSV-1-infected cells. Phosphorylation of 3H-dThd by the EHV-1 TK was inhibited by AraT, IdUrd, BVDU, and DHPG. The EHV-1 TK phosphorylated 125I-dCyd and 3H-ACV. The results indicate that EHV-1 encodes a pyrimidine deoxyribonucleoside kinase with broad nucleoside substrate specificity. These observations suggest that the failure of BVDU to inhibit EHV-1 replication is not attributable to an inability of the EHV-1 TK to phosphorylate BVDU, but may result from the incapacity of the viral TK to convert BVDU monophosphate to the triphosphate or from lack of inhibitory effect of BVDU triphosphate on viral DNA polymerase reactions.
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PMID:Phosphorylation of nucleoside analogs by equine herpesvirus type 1 pyrimidine deoxyribonucleoside kinase. 302 47

The mutagenicity of the DNA base O-alkylation adduct, O4-ethylthymine, specifically incorporated into the plasmid vector pUC8 at the unique SalI and HincII recognition sites, was studied in vivo. Escherichia coli, Micrococcus luteus and AMV DNA polymerases catalyze the incorporation of O4-ethylTMP against template adenine and guanine residues, resulting in DNA sequence alteration during subsequent replication in the host E. coli K-12 strain JM83. The greatest mutation frequency was observed with error-prone AMV DNA polymerase. High levels of cognate restriction endonuclease-resistant mutant plasmid isolates were obtained by gap replication repair in the presence of O4-ethylTTP. The yields of mutant isolates were dependent upon the relative concentration of the competing pyrimidine deoxynucleoside triphosphates, TTP and dCTP, in the misreplication reaction. Repair of incorporated O4-ethylTMP of plasmid DNA by in vitro treatment with specific alkyltransferase, prior to transformation in the host, effectively increases the mutagenic efficiency of the adduct. The results obtained are consistent with the high miscoding potential O4-ethylthymine observed in in vitro studies and its ability to base-pair with noncomplementary guanine residues in DNA.
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PMID:Site-specific gap-misrepair mutagenesis by O4-ethylthymine. 302 82

1-beta-D-Arabinofuranosyl-E-5-bromovinyluracil (BVaraU), 1-beta-D-arabinofuranosyl-E-5-iodovinyluracil (IVaraU), 1-beta-D-arabinofuranosyl-E-5-chlorovinyluracil (CVaraU) and 1-beta-D-arabinofuranosyl-5-vinyluracil (VaraU) were examined for antiviral activity against salmon herpesvirus, Oncorhynchus masou virus (OMV) in vitro using Yamame (Oncorhynchus masou) kidney cells (YNK). BVaraU, IVaraU, CVaraU and VaraU were highly active against OMV; 50% inhibitory concentration (IC50): 0.01, 0.003, 0.003, 0.003 microgram/ml, respectively. The IC50 of 5-bromovinyl-2'-deoxyuridine (BVDU) was 0.3 microgram/ml. The lower activity may be due to cleavage of it N-glycosyl linkage by pyrimidine nucleoside phosphorylases (i.e. thymidine phosphorylase) during the incubation period. The arabinofuranosyl counterparts are resistant to this (these) enzyme(s). Both OMV-induced DNA polymerase and cellular DNA polymerase alpha were strongly inhibited by BVaraU 5'-triphosphate (BVaraUTP). In an in vivo study, daily immersion of OMV-infected chum salmon (Oncorhynchus keta) fry into aqueous solution of BVaraU (5 micrograms/ml, 30 min/day, 30 times) did not increase the life span of infected fish.
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PMID:Antiviral activity of various 1-beta-D-arabinofuranosyl-E-5-halogenovinyluracils and E-5-bromovinyl-2'-deoxyuridine against salmon herpes virus, Oncorhynchus masou virus (OMV). 303 48

The formation of pyridine-pyrimidine- and pyrimidine-pyrimidine base pairs after in vitro DNA replication with the large fragment of Escherichia coli DNA polymerase I indicates that Watson-Crick-like base pairing between pyrimidine bases can occur in the enzyme due to the presence of the rare tautomers of deoxycytidylate and thymidylate in the template strand. The implications to mispair formation in DNA, such as the difference between the structures of the mispairs during and after replication, are discussed and the possible action of mutagenic DNA protonating and deprotonating agents in vivo is considered.
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PMID:Mispair formation in DNA can involve rare tautomeric forms in the template. 305 6

The role of exonuclease activity in trans-lesion DNA replication with Escherichia coli DNA polymerase III holoenzyme was investigated. RecA protein inhibited the 3'----5' exonuclease activity of the polymerase 2-fold when assayed in the absence of replication and had no effect on turnover of dNTPs into dNMPs. In contrast, single-stranded DNA-binding protein, which had no effect on the exonuclease activity in the absence of replication, showed a pronounced 7-fold suppression of the 3'----5' exonuclease activity during replication. The excision of incorporated dNMP alpha S residues from DNA by the 3'----5' exonuclease activity of DNA polymerase III holoenzyme was inhibited 10-20-fold; still no increase in bypass of pyrimidine photodimers was observed. Thus, in agreement with our previous results in which the exonuclease activity was inhibited at the protein level (Livneh, Z. (1986) J. Biol. Chem. 261, 9526-9533), inhibition at the DNA level also did not increase bypass of photodimers. Fractionation of the replication mixture after termination of DNA synthesis on a Bio-Gel A-5m column under conditions which favor polymerase-DNA binding yielded a termination complex which could perform turnover of dNTPs into dNMPs. Adding challenge-primed single-stranded DNA to the complex yielded a burst of DNA synthesis which was promoted most likely by DNA polymerase III holoenzyme molecules transferred from the termination complex to the challenge DNA thus demonstrating the instability of the polymerase-DNA association. Addition of a fresh sample of DNA polymerase III holoenzyme to purified termination products, which consist primarily of partially replicated molecules with nascent chains terminated at UV lesions, did not result in any net DNA synthesis as expected. However, reactivation of lesion-terminated primers was achieved by pretreatment with a 3'----5' exonuclease which excised 200 nucleotides or more, generating new 3'-OH termini located away from the UV lesions. When these exonuclease-treated products were subjected to a second round of replication, an increased level of DNA synthesis was observed including additional bypass of photodimers. These results suggest the possibility that 3'----5' exonuclease processing might be required at least transiently during one of the stages of trans-lesion DNA replication, which is believed to be the mechanism of SOS-targeted mutagenesis.
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PMID:The role of exonucleolytic processing and polymerase-DNA association in bypass of lesions during replication in vitro. Significance for SOS-targeted mutagenesis. 305 41

Substituent effects governing inhibition of DNA polymerase III from Bacillus subtilis were examined in several series of N6-substituted 6-aminopyrimidines. The presence of alkyl groups as large as n-butyl in the 3-position of 6-(5-indanylamino)uracil had no effect on inhibitor-enzyme binding. Substituents in the 4-position of a series of 2-amino-6-(benzylamino)pyrimidines had complex effects: alkoxy and phenoxy derivatives were less active than the parent 4-oxo (isocytosine) compound, but alkylphenoxy and halophenoxy derivatives were more active than the 4-phenoxy compound itself, suggesting that hydrophobic binding can occur between 4-substitutents and the enzyme surface and that space between the pyrimidine ring and pol III may represent the active site of the enzyme. Replacement of 5-H by methyl and ethyl groups drastically decreased inhibitory activity of 6-(benzylamino)- and 6-p-toluidinouracils, but 5-bromo and 5-iodo analogues were equipotent with the parent compounds. These results indicate that the phenyl rings of these compounds must exist in conformations in which they are perpendicular to the pyrimidine ring plane and that charge-transfer stabilization of such "active conformations" may compensate for steric barriers from 5-halo groups in the inhibitor-enzyme complex.
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PMID:Inhibitors of Bacillus subtilis DNA polymerase III. Influence of modifications in the pyrimidine ring of anilino- and (benzylamino)pyrimidines. 308 85

The authors' approach to the design of DNA polymerase-specific inhibitors, an approach based on the mechanism of action of 6-(p-hydroxyphenylazo)uracil, has been to disguise nucleic acid bases to mimic the purine substrates dGTP and dATP. Specifically, the strategy has been to synthesize bases with substituents that endow them with the capacity: to seek and react with unique features of the active site of a polymerase; and to form H bonds with complementary template pyrimidines apposing the active site. This strategy has yielded a series of novel, enzyme-specific dATP and dGTP analogues which are non-polymerizable and which inhibit their target polymerase by sequestering it to a complementary pyrimidine residue in primer:template. The work has involved primarily two replication-specific polymerases, B. subtilis DNA polymerase III (pol III) and mammalian DNA polymerase alpha (pol alpha). The initial design exploited the pyrimidine nucleus and produced inhibitors with Ki values in the micromolar range. Principles established with the pyrimidine derivatives have led to the development of bona fide purine nucleotide analogues which act as DNA polymerase inhibitors of high selectivity and unprecedented potency. For example, BuPdGTP, the 2'-deoxyribonucleoside 5'-triphosphate of N2-(p-n-butylphenyl)guanine (BuPG), lacks discernible activity against mammalian polymerases beta and gamma, whereas it inhibits mammalian pol alpha with a Ki of less than 10 nanomolar. Currently, the authors are exploiting BuPdGTP, BuPdGDP, and similar butylanilino derivatives of dATP to probe the active site of pol alpha and to develop other N2-substituted analogues which can bind selectively to the substrate sites of other important polymerases and nucleotide binding proteins.
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PMID:Rational design of substrate analogues targeted to selectively inhibit replication-specific DNA polymerases. 309 44

Those pyrimidine dimers that are repaired in confluent xeroderma pigmentosum Group C cells are clustered together in the genome. Although the average level of repair in this complementation group is of the order of 25% of normal, this percentage represents normal levels of repair in one quarter of the genome and little repair in the remainder. The factors that regulate this clustering process have been investigated using inhibitors of the initial incision step of repair (novobiocin) and of the polymerization step (aphidicolin). Novobiocin at a concentration that permitted 30% of repair to continue reduced the clustering of mended sites only slightly. Aphidicolin, in contrast, at a concentration that permitted 30 to 60% of repair to continue caused the mended sites to be distributed randomly. The clustering of repair sites seen in xeroderma pigmentosum Group C cells, therefore, is produced by an excision repair mechanism in which an aphidicolin-sensitive DNA polymerase, presumably alpha, plays an important regulatory role in determining which damaged sites are mended.
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PMID:Relative importance of incision and polymerase activities in determining the distribution of damaged sites that are mended in xeroderma pigmentosum group C cells. 310 77

The bimodal-incision nature of the reaction of UV-irradiated DNA catalyzed by the Escherichia coli uvrABC protein complex potentially leads to excision of a 12- to 13-nucleotide-long damaged fragment. However, the oligonucleotide fragment containing the UV-induced pyrimidine dimer is not released under nondenaturing in vitro reaction conditions. Also, the uvrABC proteins are stably bound to the incised DNA and do not turn over after the incision event. In this communication it is shown that release of the damaged fragment from the parental uvrABC-incised DNA is dependent upon either chelating conditions or the simultaneous addition of the uvrD gene product (helicase II) and the polA gene product (DNA polymerase I) when polymerization of deoxynucleoside triphosphate substrates is concomitantly catalyzed. The product of this multiprotein-catalyzed series of reactions serves as a substrate for polynucleotide ligase, resulting in the restoration of the integrity of the strands of DNA. The addition of the uvrD protein to the incised DNA-uvrABC complex also results in turnover of the uvrC protein. It is suggested that the repair processes of incision, excision, resynthesis, and ligation are coordinately catalyzed by a complex of proteins in a "repairosome" configuration.
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PMID:Involvement of helicase II (uvrD gene product) and DNA polymerase I in excision mediated by the uvrABC protein complex. 316 Oct 77


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