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)

The treatment of tamoxifen, widely used as adjuvant chemotherapy for breast cancer, increases significantly the risk of developing endometrial cancer. The miscoding properties of tamoxifen-derived DNA adducts, alpha-(N2-deoxyguanosinyl)tamoxifens (dG-N2-tamoxifen), have been explored, using an in vitro experimental system to quantify base substitutions and deletions. Site-specifically modified oligodeoxynucleotides containing an epimer of trans- and cis-forms of dG-N2-tamoxifens were prepared postsynthetically and used as templates in primer extension reactions catalyzed by mammalian DNA polymerases alpha, beta, and delta. Pol alpha catalyzed incorporation of dCMP and dAMP opposite all four stereoisomers of dG-N2-tamoxifen, accompanied by lesser amounts of dGMP. In contrast, pol delta catalyzed preferential incorporation of dCMP, a correct base, opposite the lesions; one of the trans-forms of dG-N2-tamoxifens only promoted incorporation of dTMP. Using pol beta, preferential incorporation of dCMP, along with small amounts of incorporation of dAMP and dGMP, was detected. One- and two base deletions were also observed with pol alpha and pol beta. The miscoding specificities and frequencies of dG-N2-tamoxifens varied depending on the DNA polymerase used. In addition, with pol alpha and pol beta, large amounts of 5-base deletions were preferentially formed at the cis-forms of dG-N2-tamoxifen, but not at the trans-forms of dG-N2-tamoxifen. We conclude that dG-N2-tamoxifen adducts have high miscoding potentials.
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PMID:Miscoding potential of tamoxifen-derived DNA adducts: alpha-(N2-deoxyguanosinyl)tamoxifen. 933 62

The fidelity of Escherichia coli DNA polymerase III (pol III) is measured and the effects of beta, gamma processivity and epsilon proofreading subunits are evaluated using a gel kinetic assay. Pol III holoenzyme synthesizes DNA with extremely high fidelity, misincorporating dTMP, dAMP, and dGMP opposite a template G target with efficiencies finc = 5.6 x 10(-6), 4.2 x 10(-7), and 7 x 10(-7), respectively. Elevated dGMP.G and dTMP.G misincorporation efficiencies of 3.2 x 10(-5) and 5.8 x 10(-4), attributed to a "dNTP-stabilized" DNA misalignment mechanism, occur when C and A, respectively, are located one base downstream from the template target G. At least 92% of misinserted nucleotides are excised by pol III holoenzyme in the absence of a next correct "rescue" nucleotide. As rescue dNTP concentrations are increased, pol III holoenzyme suffers a maximum 8-fold reduction in fidelity as proofreading of mispaired primer termini are reduced in competition with incorporation of a next correct nucleotide. Compared with pol III holoenzyme, the alpha holoenzyme, which cannot proofread, has 47-, 32-, and 13-fold higher misincorporation rates for dGMP.G, dTMP.G, and dAMP.G mispairs. Both the beta, gamma complex and the downstream nucleotide have little effect on the fidelity of catalytic alpha subunit. An analysis of the gel kinetic fidelity assay when multiple polymerase-DNA encounters occur is presented in the "Appendix" (see Fygenson, D. K., and Goodman, M. F. (1997) J. Biol. Chem. 272, 27931-27935 (accompanying paper)).
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PMID:Fidelity of Escherichia coli DNA polymerase III holoenzyme. The effects of beta, gamma complex processivity proteins and epsilon proofreading exonuclease on nucleotide misincorporation efficiencies. 934 41

Aryl radicals and arenediazonium ions are suspected to react with cellular DNA, resulting in C8-arylguanine adducts. 8-Phenyl-2'-deoxyguanosine (8-PhdG) was synthesized as a model adduct by reacting dG with benzenediazonium chloride and incorporated into oligodeoxynucleotides using phosphoramidite techniques. A site-specifically modified oligodeoxynucleotide containing a single 8-PhdG was then used as a template for primer extension reactions catalyzed by the intact (exo+) or 3'-->5' exonuclease-free (exo-) Klenow fragment of Escherichia coli DNA polymerase I and mammalian DNA polymerase alpha (pol alpha). Although primer extensions catalyzed by the Klenow fragments were retarded at the position of 8-PhdG, most of the primer extension passed the lesion to form the fully extended products. In contrast, primer extensions catalyzed by pol alpha were strongly blocked opposite the lesion. The fully extended products formed during DNA synthesis were analyzed to quantify the miscoding specificities of 8-PhdG. The exo- Klenow fragment incorporated primarily dCMP, the correct base, opposite 8-PhdG, along with small amounts of incorporation of dAMP. Two-base deletions were also observed. In contrast, the exo+ Klenow fragment incorporated dCMP opposite the lesion. When pol alpha was used, 8-PhdG promoted small amounts of misincorporation of dAMP and dGMP as well as one- and two-base deletions. The duplex containing 8-PhdG.dG was thermally and thermodynamically more stable than dG.dG. The duplex containing 8-PhdG.dA was thermodynamically more stable than dG.dA. We conclude that 8-PhdG is a weak miscoding lesion, capable of generating G-->T and G-->C transversions and deletions in cells.
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PMID:Synthesis and miscoding specificity of oligodeoxynucleotide containing 8-phenyl-2'-deoxyguanosine. 943 25

To investigate whether or not DNA polymerases alpha, delta, and epsilon from tumor cells have acquired properties that might be responsible for mutations found in tumor development, we investigated copying fidelities of DNA polymerases alpha, delta, and epsilon from the highly malignant Novikoff hepatoma cells and compared them to the corresponding enzymes from normal rat liver. DNA polymerases were purified more than 300-fold by three chromatographic steps. Copying fidelity was studied using steady-state kinetics and an 18-mer oligonucleotide primed with a 12-mer (13-mer for extension experiments) as DNA primer-template. Three experimental approaches were chosen: i) extension of DNA primers with mismatched 3'-OH ends opposite dGMP, ii) DNA insertion of nucleotides opposite m6G in the template and iii) extension of DNA primers with mismatched 3'-OH ends opposite m6G. i) Extension of DNA primers with mismatched 3'-OH ends opposite dGMP. DNA primer templates containing G:T and G:A mispairs at the 3'-OH position of the primer were easily extended by DNA polymerases alpha, delta and epsilon from both normal rat liver and Novikoff hepatoma cells. The G:G mismatch was elongated with low efficiency. Notably, DNA polymerase alpha from Novikoff hepatoma cells extended G:A and G:G mismatches significantly faster than the enzyme from normal cells. ii) Insertion of nucleotides opposite m6G. DNA polymerases alpha, delta, and epsilon from normal rat liver preferably catalyzed incorporation of dAMP opposite m6G at dNTP concentrations < 100 microM. When dNTP concentrations were raised to > or = 100 microM, dCMP (DNA polymerases delta and epsilon) and dTMP (DNA polymerase alpha) were also incorporated. The same insertion characteristics were found for the enzymes from Novikoff cells, however, insertion efficiencies of dAMP and dCMP were significantly higher for polymerases delta and epsilon. iii) Extension of primers with mismatched 3'-OH ends opposite m6G. Only m6G:dAMP and m6G:dCMP mismatches were extended by DNA polymerases alpha, delta and epsilon from both sources. No differences in extension efficiency were observed between the enzymes from normal and hepatoma cells. Taken together, our results suggest that DNA polymerases alpha, delta, and epsilon from Novikoff cells catalyzed incorporation of the wrong nucleotides more readily and extended mismatches more easily. These results may provide a rationale why numerous mutations accumulate during tumor development.
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PMID:Evidence for reduced copying fidelity of DNA polymerases alpha, delta, and epsilon from Novikoff hepatoma cells. 962 Feb 26

PMEG (9-(2-phosphonylmethoxyethyl)guanine) is an acyclic nucleotide analog being evaluated for its anti-proliferative activity. We examined the inhibitory effects of PMEG diphosphate (PMEGpp) toward DNA polymerases (pol) delta and epsilon and found it to be a competitive inhibitor of both these enzymes. The apparent Ki values for PMEGpp were 3-4 times lower than the Km values for dGTP. The analog was shown to function as a substrate and to be incorporated into DNA by both enzymes. Examination of the ability of pol delta and pol epsilon to repair the incorporated PMEG revealed that pol epsilon could elongate PMEG-terminated primers in both matched and mismatched positions with an efficiency equal to 27 and 85% that observed for dGMP-terminated control template-primers. Because PMEG acts as an absolute DNA chain terminator, the elongation of PMEG-terminated primers is possible only by cooperation of the 3'-5'-exonuclease and DNA polymerase activities of the enzyme. In contrast to pol epsilon, pol delta exhibited negligible activity on these template-primers, indicating that pol epsilon, but not pol delta, can repair the incorporated analog.
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PMID:Incorporation and excision of 9-(2-phosphonylmethoxyethyl)guanine (PMEG) by DNA polymerase delta and epsilon in vitro. 970 37

Oxidative damage to DNA bases commonly resultsin the formation of oxidized purines, particularly 7,8-dihydro-8-oxoguanine (8-oxoG) and 7,8-dihydro-8-oxoadenine (8-oxoA), the former being a well-known mutagenic lesion. Since 8-oxoG is readily subject to further oxidation compared with normal bases, the insertion of a base during DNA synthesis opposite an oxidized form of 8-oxoG was investigated in vitro. A synthetic template containing a single 8-oxoG lesion was first treated with different one-electron oxidants or under singlet oxygen conditions and then subjected to primer extension catalyzed by Klenow fragment exo- (Kf exo-), calf thymus DNA polymerase alpha (pol alpha) or human DNA polymerase beta (pol beta). Consistent with previous reports, dAMP and dCMP are inserted selectively opposite 8-oxoG with all three DNA polymerases. Interestingly, oxidation of 8-oxoG was found to induce dAMP and dGMP insertion opposite the lesion by Kf exo- with transient inhibition of primer extension occurring at the site of the modified base. Furthermore, the lesion constitutes a block during DNA synthesis by pol alpha and pol beta. Experiments with an 8-oxoA-modified template oligonucleotide show that both 8-oxoA and an oxidized form of 8-oxoA direct insertion of dTMP by Kf exo-. Mass spectrometric analysis of 8-oxoG-containing oligonucleotides before and after oxidation with IrCl62-are consistent with oxidation of primarily the 8-oxoG site, resulting in formation of a guanidinohydantoin moiety as the major product. No evidence for formation of abasic sites was obtained. These results demonstrate that an oxidized form of 8-oxoG, possibly guanidinohydantoin, may direct misreading and misinsertion of dNTPs during DNA synthesis. If such a process occurred in vivo, it would represent a point mutagenic lesion leading to G-->T and G-->C transversions. However, the corresponding oxidized form of 8-oxoA primarily shows correct insertion of T during DNA synthesis with Kf exo-.
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PMID:Insertion of dGMP and dAMP during in vitro DNA synthesis opposite an oxidized form of 7,8-dihydro-8-oxoguanine. 986 71

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a common oxidative DNA lesion, favors a syn-conformation in DNA, enabling formation of stable 8-oxo-dG.A base mispairs resulting in G.C --> T.A transversion mutations. When human DNA polymerase (pol) beta was used to copy a short single-stranded gap containing a site-directed 8-oxo-dG lesion, incorporation of dAMP opposite 8-oxo-dG was slightly favored over dCMP depending on "downstream" sequence context. Unexpectedly, however, a significant increase in dCMP.A and dGMP.A mispairs was also observed at the "upstream" 3'-template site adjacent to the lesion. Errors at these undamaged template sites occurred in four sequence contexts with both gapped and primed single-stranded DNA templates, but not when pol alpha replaced pol beta. Error rates at sites adjacent to 8-oxo-dG were roughly 1% of the values opposite 8-oxo-dG, potentially generating tandem mutations during in vivo short-gap repair synthesis by pol beta. When 8-oxo-dG was replaced with 8-bromo-2'-deoxyguanosine, incorporation of dCMP was strongly favored by both enzymes, with no detectable misincorporation occurring at neighboring template sites.
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PMID:"Action-at-a-distance" mutagenesis. 8-oxo-7, 8-dihydro-2'-deoxyguanosine causes base substitution errors at neighboring template sites when copied by DNA polymerase beta. 1033 98

To examine the hypothesis that interactions between a DNA polymerase and the DNA minor groove are critical for accurate DNA synthesis, we studied the fidelity of DNA polymerase beta mutants at residue Arg(283), where arginine, which interacts with the minor groove at the active site, is replaced by alanine or lysine. Alanine substitution, removing minor groove interactions, strongly reduces polymerase selectivity for all single-base mispairs examined. In contrast, the lysine substitution, which retains significant interactions with the minor groove, has wild-type-like selectivity for T.dGMP and A.dGMP mispairs but reduced selectivity for T.dCMP and A.dCMP mispairs. Examination of DNA crystal structures of these four mispairs indicates that the two mispairs excluded by the lysine mutant have an atom (N2) in an unfavorable position in the minor groove, while the two mispairs permitted by the lysine mutant do not. These results suggest that unfavorable interactions between an active site amino acid side chain and mispair-specific atoms in the minor groove contribute to DNA polymerase specificity.
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PMID:Base substitution specificity of DNA polymerase beta depends on interactions in the DNA minor groove. 1040 11

Replication of DNA lesions leads to the formation of mutations. In Escherichia coli this process is regulated by the SOS stress response, and requires the mutagenesis proteins UmuC and UmuD'. Analysis of translesion replication using a recently reconstituted in vitro system (Reuven, N. B., Tomer, G., and Livneh, Z. (1998) Mol. Cell 2, 191-199) revealed that lesion bypass occurred with a UmuC fusion protein, UmuD', RecA, and SSB in the absence of added DNA polymerase. Further analysis revealed that UmuC was a DNA polymerase (E. coli DNA polymerase V), with a weak polymerizing activity. Upon addition of UmuD', RecA, and SSB, the UmuC DNA polymerase was greatly activated, and replicated a synthetic abasic site with great efficiency (45% bypass in 6 min), 10-100-fold higher than E. coli DNA polymerases I, II, or III holoenzyme. Analysis of bypass products revealed insertion of primarily dAMP (69%), and to a lesser degree dGMP (31%) opposite the abasic site. The UmuC104 mutant protein was defective both in lesion bypass and in DNA synthesis. These results indicate that UmuC is a UmuD'-, RecA-, and SSB-activated DNA polymerase, which is specialized for lesion bypass. UmuC is a member of a new family of DNA polymerases which are specialized for lesion bypass, and include the yeast RAD30 and the human XP-V genes, encoding DNA polymerase eta.
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PMID:The mutagenesis protein UmuC is a DNA polymerase activated by UmuD', RecA, and SSB and is specialized for translesion replication. 1054 96

3,N(4)-Ethano-2'-deoxycytidine (ethano-dC) may be incorporated successfully into synthetic oligodeoxynucleotides by omitting the capping procedure used in the automated DNA synthetic protocols immediately after inserting the lesion and in all iterations thereafter. Ethano-dC is sensitive to acetic anhydride found in the capping reagent, and multiple oligomeric products are formed. These products were identified by examining the reaction of ethano-dC with the capping reagent, and several acetylated, ring-opened products were characterized by electrospray mass spectrometry and collision induced dissociation experiments on a tandem quadrupole mass spectrometer. A scheme for the formation of the acetylated products is proposed. In addition, the mutagenic profile of ethano-dC was re-examined and compared to that for etheno-dC. Ethano-dC is principally a blocking lesion; however, when encountered by the exo(-)Klenow fragment of DNA polymerase, dAMP (22%), TMP (16%), dGMP (5.3%) and dCMP (1.2%) were all incorporated opposite ethano-dC, along with an oligomer containing a one-base deletion (0.6%).
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PMID:3,N(4)-ethano-2'-deoxycytidine: chemistry of incorporation into oligomeric DNA and reassessment of miscoding potential. 1057 72


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