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)

We have investigated the mechanism of frameshift (deletion) mutagenesis induced by acetylaminofluorene- (AAF-) derived DNA adducts. dG-AAF-modified oligodeoxynucleotides, with different bases positioned 5' to the lesion, were annealed to (32)P-labeled 13-mer primers and then used in primer extension reactions catalyzed by the 3'-->5' exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I. When the dNMP positioned opposite dG-AAF could pair with its complementary base at the 5' flanking position, single-base deletions were produced at high frequency. Similarly, when the complementary base was two positions 5' to the dG-AAF, two-base deletions occurred. The relative frequency of base insertions opposite dG-AAF followed the order dCMP > dAMP > dGMP > dTMP; the frequency of dNTP insertion opposite the lesion paralleled the formation of frameshift deletions. When a template designed to induce three-base deletions was used for translesion synthesis catalyzed by the exo(-) Klenow fragment, the expected three-base deletion was formed. When dG-AAF-modified templates containing iterated bases 5' to the lesion were annealed to primers with the complementary dNMP positioned opposite the lesion, the dNMP inserted opposite the dG-AAF tended to pair with the complementary base 5' to the lesion, thereby forming shorter deletions. Taken together, these results support the molecular mechanism for frameshift deletion proposed earlier by Shibutani and Grollman in which direct base insertion precedes misalignment [(1993) J. Biol. Chem. 268, 11703].
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PMID:Mechanism of frameshift (deletion) generated by acetylaminofluorene-derived DNA adducts in vitro. 1559 49

It has been suggested that carcinogenesis associated with chronic inflammation involves DNA damage by nitric oxide (NO) and other reactive species secreted from macrophages and neutrophils. The guanine moiety of DNA reacts with NO, yielding two major deamination products: xanthine (Xan) and oxanine (Oxa). Oxa reacts further with polyamines and DNA binding proteins to form cross-link adducts. In the present study, we characterized the structure of the cross-link adducts of Oxa with spermine (Oxa-Sp). Spectrometric analysis of Oxa-Sp adducts showed that they are ring-opened adducts of Oxa covalently bonded to the terminal amino (major product) and internal imino (minor product) groups of spermine. To assess genotoxic potential, Xan, Oxa, Oxa-Sp and an abasic (AP) site were site specifically incorporated into oligonucleotide templates. These lesions differentially blocked in vitro DNA synthesis catalyzed by DNA polymerase I Klenow fragment (Pol I Kf). The relative efficiency of translesion synthesis was G (1) > Oxa (0.19) > Xan (0.12) > AP (0.088) > Oxa-Sp (0.035). Primer extension assays with a single nucleotide and Pol I Kf revealed that non-mutagenic dCMP was inserted most efficiently opposite Xan and Oxa, with the extent of primer elongation being 65% for Xan and 68% for Oxa. However, mutagenic nucleotides were also inserted. The extent of primer elongation for Xan was 16% with dTMP and 14% with dGMP, whereas that for Oxa was 49% with dTMP. For Oxa-Sp, mutagenic dAMP (13%) was preferentially inserted. Accordingly, when generated in vivo, Xan and Oxa would constitute moderate blocks to DNA synthesis and primarily elicit G:C to A:T transitions when bypassed, whereas Oxa-Sp would strongly block DNA synthesis and elicit G:C to T:A transversions.
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PMID:Assessment of the genotoxic potential of nitric oxide-induced guanine lesions by in vitro reactions with Escherichia coli DNA polymerase I. 1584 89

We recently identified, from the gamma-irradiation mixture of duplex DNA, a new intrastrand G[8-5]C cross-link lesion, in which the C8 atom of guanine and the C5 atom of its 3' neighboring cytosine are covalently bonded, and carried out in vitro replication studies for the lesion-bearing substrate with a translesion synthesis polymerase, yeast polymerase eta. Here we extended the in vitro replication studies to two replicative polymerases, exonuclease-deficient bacteriophage T7 DNA polymerase (T7(-)) and HIV reverse transcriptase (HIV-RT). Primer extension assays showed that both polymerases stopped synthesis after incorporating a nucleotide opposite the 3'-cytosine in the G[8-5]C lesion. Steady-state kinetic measurements for nucleotide incorporation opposite the 3'-cytosine of the lesion showed that both T7(-) and HIV-RT preferentially incorporated the correct nucleotide, dGMP. We also examined the thermal stabilities and base pairing properties of G[8-5]C in d(ATGGCG[8-5]CGCTAT). The G[8-5]C lesion destabilizes the duplex form by approximately 4 kcal/mol in free energy at 25 degrees C relative to the undamaged parent duplex, and the thermally most stable duplex has natural bases opposite the lesion.
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PMID:Thermodynamic and in vitro replication studies of an intrastrand G[8-5]C cross-link lesion. 1595 95

Clustered DNA damages are well-established characteristics of ionizing radiation. As a model clustered lesion in the same strand of DNA, we have evaluated the mutagenic potential of 8-oxoguanine (8-oxoG) adjacent to a uracil in simian kidney cells using a phagemid vector. The uracil residue would be excised by the enzyme uracil DNA glycosylase in vivo generating an abasic site (AP site). A solitary uracil in either GUGTC or GTGUC sequence context provided >60% progeny containing GTGTC indicating that dAMP incorporation opposite the AP site or uracil occurred, but a >30% population showed replacement of U by A, C, or G, which suggests that dTMP, dGMP, or dCMP incorporation also occurred, respectively, opposite the AP site. While the preference for targeted base substitutions at the GUG site was T >> C > A > G, the same at the GUC site was T >> A > C > G. We conclude that base incorporation opposite an AP site is sequence-dependent. For 8-oxoG, as compared to 23-24% G-->T mutants from a single 8-oxoG in a TG(8-oxo)T sequence context, the tandem lesions UG(8-oxo)T and TG(8-oxo)U generated approximately 60 and >85% progeny, respectively, that did not contain the TGT sequence. A significant fraction of tandem mutations were detected when uracil was adjacent to 8-oxoG. What we found most interesting is that the total targeted G(8-oxo)-->T transversions that included both single and tandem mutations at the TG(8-oxo)U site was nearly 60% relative to about 30% at the UG(8-oxo)T site. A higher mutational frequency at the TG(8-oxo)U sequence may arise from a change in DNA polymerase that is more error prone. Thermal melting experiments showed that the Tm for the 8-oxoG:C pair in the TG(8-oxo)(AP*) sequence in a 12-mer was lower than the same in a (AP*)G(8-oxo)T 12-mer with deltadeltaG 0.8 kcal/mol (where AP* represents tetrahydrofuran, the model abasic site). When the 8-oxoG:C pair in each sequence was compared with a 8-oxoG:A pair, the former was found to be more stable than the latter. The preference for C over A opposite 8-oxoG for the (AP*)G(8-oxo)T 12-mer duplex with a deltadeltaG of 1.6 kcal/mol dropped to 0.4 kcal/mol in the TG(8-oxo)(AP*) 12-mer duplex. This suggests that the polymerase discrimination to incorporate dCMP over dAMP would be less efficient in the TG(8-oxo)(AP*) sequence relative to (AP*)G(8-oxo)T. Additionally, the efficiency of recognition and excision of A opposite 8-oxoG by a mismatch repair protein may be impaired in the TG(8-oxo)(AP*) sequence context.
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PMID:Mutagenesis of 8-oxoguanine adjacent to an abasic site in simian kidney cells: tandem mutations and enhancement of G-->T transversions. 1609 91

The translesion synthesis (TLS) capacity of the thermostable DNA polymerases Taq, Tte and Tte-seq utilizing a synthetic abasic site, tetrahydrofuran (THF), and an 8-oxoguanine-containing DNA template was investigated. Measurements with human DNA polymerase beta were used as a "positive control". Thermostable DNA polymerases were observed to perform TLS with different specificities on both substrates. With a THF-containing template, dGMP was preferentially inserted by all the DNA polymerases. In the presence of Mn(II) as a cofactor, all the polymerases incorporated dCMP opposite 8-oxoguanine whereas, in the presence of Mg(II) ions, dAMP was incorporated. It was found that none of the thermophilic DNA polymerases utilized dTTP with either an 8-oxoguanine or a THF-containing template. In all cases, DNA duplex containing THF as damage was processed to full length less effectively than DNA duplex containing 8-oxoguanine.
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PMID:Thermostable DNA polymerases can perform translesion synthesis using 8-oxoguanine and tetrahydrofuran-containing DNA templates. 1633 85

7,8-Dihydro-8-oxoguanine (8-oxo-Gua) and its nucleoside in cytosol are derived from the repair of oxidative DNA and the cleanup of oxidatively damaged DNA precursors, respectively. While the harmful effects of 8-oxo-Gua present in DNA have been studied extensively, few have reported its effects on cytosolic function. Our previous study showed that the addition of 8-oxo-dG to culture media caused an accumulation of 8-oxo-Gua in nuclear DNA in several leukemic cells including KG-1, which lack 8-oxoguanine glycosylase 1 (OGG1) activity due to mutational loss. However, the mechanism underlying 8-oxo-Gua level increases in DNA has not been addressed. In this study, we elucidated the metabolic fate of 8-oxo-Gua-containing nucleotide and the effect of exogenous 8-oxo-dG on DNA synthesis in KG-1 cells. We found that 8-oxo-dGMP was rapidly dephosphorylated to 8-oxo-dG rather than phosphorylated to 8-oxo-dGDP, thus indicating that 8-oxo-Gua-containing molecule is not used as a substrate for DNA synthesis in KG-1 cells. In fact, radiolabeled 8-oxo-dG was incubated but radioactivity was not detected in nuclear DNA of KG-1 cells, showing that 8-oxo-dG is not directly incorporated into DNA. Interestingly, the activity of DNA polymerase beta, which synthesize DNA with low fidelity increased in KG-1 cells treated with 8-oxo-dG, whereas the expression of DNA polymerase alpha decreased. In addition, the accumulation of 8-oxo-Gua in KG-1 DNA was completely inhibited by a specific inhibitor of DNA polymerase beta. Thus, our findings address that the insertion of 8-oxo-dG into KG-1 DNA is not due to the direct incorporation of exogenous 8-oxo-dG, but rather to the inaccurate incorporation of endogenous 8-oxo-dGTP by DNA polymerase beta. It further suggests that 8-oxo-dG in the cytosol may function as an active molecule itself and perturb the well-defined DNA synthesis.
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PMID:Exogenous 8-oxo-dG is not utilized for nucleotide synthesis but enhances the accumulation of 8-oxo-Gua in DNA through error-prone DNA synthesis. 1647 28

Cyclobutane pyrimidine dimers (CPDs) are responsible for a considerable fraction of sunlight-induced C to T and 5-methycytosine (mC) to T mutations in mammalian cells, though the precise mechanism is unknown. One possibility is that the C or mC of a CPD is not mutagenic and must first deaminate to U or T, respectively, for A to be inserted by a DNA polymerase. Alternatively, A might be directly inserted opposite the C or mC prior to deamination via an E-imino tautomer of the C or mC or by a nontemplated mechanism in which the photoproduct is sterically excluded from the active site. We have taken advantage of the retarding effect of C5 methylation on the deamination rate of cis-syn-cyclobutane dimers to prepare a template containing the cis-syn-cyclobutane dimer of mCT. Through the use of single-hit and multiple-hit competition assays, the catalytic core of pol eta was found to insert dGMP opposite the mC of the CPD with about a 120:1 selectivity relative to dAMP. No significant insertion of dTTP or dCMP was detected. The high fidelity of nonmutagenic insertion opposite the mC of the CPD provides strong support for the deamination-bypass mechanism for the origin of sunlight induced C --> T mutations.
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PMID:DNA synthesis past a 5-methylC-containing cis-syn-cyclobutane pyrimidine dimer by yeast pol eta is highly nonmutagenic. 1686 79

Pseudomonas aeruginosa is a human opportunistic pathogen that chronically infects the lungs of cystic fibrosis patients and is the leading cause of morbidity and mortality of people afflicted with this disease. A striking correlation between mutagenesis and the persistence of P. aeruginosa has been reported. In other well-studied organisms, error-prone replication by Y family DNA polymerases contributes significantly to mutagenesis. Based on an analysis of the PAO1 genome sequence, P. aeruginosa contains a single Y family DNA polymerase encoded by the dinB gene. As part of an effort to understand the mechanisms of mutagenesis in P. aeruginosa, we have cloned the dinB gene of P. aeruginosa and utilized a combination of genetic and biochemical approaches to characterize the activity and regulation of the P. aeruginosa DinB protein (DinB(Pa)). Our results indicate that DinB(Pa) is a distributive DNA polymerase that lacks intrinsic proofreading activity in vitro. Modest overexpression of DinB(Pa) from a plasmid conferred a mutator phenotype in both Escherichia coli and P. aeruginosa. An examination of this mutator phenotype indicated that DinB(Pa) has a propensity to promote C-->A transversions and -1 frameshift mutations within poly(dGMP) and poly(dAMP) runs. The characterization of lexA+ and DeltalexA::aacC1 P. aeruginosa strains, together with in vitro DNA binding assays utilizing cell extracts or purified P. aeruginosa LexA protein (LexA(Pa)), indicated that the transcription of the dinB gene is regulated as part of an SOS-like response. The deletion of the dinB(Pa) gene sensitized P. aeruginosa to nitrofurazone and 4-nitroquinoline-1-oxide, consistent with a role for DinB(Pa) in translesion DNA synthesis over N2-dG adducts. Finally, P. aeruginosa exhibited a UV-inducible mutator phenotype that was independent of dinB(Pa) function and instead required polA and polC, which encode DNA polymerase I and the second DNA polymerase III enzyme, respectively. Possible roles of the P. aeruginosa dinB, polA, and polC gene products in mutagenesis are discussed.
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PMID:Role of Pseudomonas aeruginosa dinB-encoded DNA polymerase IV in mutagenesis. 1704 Oct 45

Methylglyoxal is a highly reactive alpha-ketoaldehyde that is produced endogenously and present in the environment and foods. It can modify DNA and proteins to form advanced glycation end products (AGEs). Emerging evidence has shown that N2-(1-carboxyethyl)-2'-deoxyguanosine (N2-CEdG) is a major marker for AGE-linked DNA adducts. Here, we report, for the first time, the preparation of oligodeoxyribonucleotides (ODNs) containing individual diastereomers of N2-CEdG via a postoligomerization synthesis method, which provided authentic substrates for examining the replication and repair of this lesion. In addition, thermodynamic parameters derived from melting temperature data revealed that the two diastereomers of N2-CEdG destabilized significantly the double helix as represented by a 4 kcal/mol increase in Gibbs free energy for duplex formation at 25 degrees C. Primer extension assay results demonstrated that both diastereomers of N2-CEdG could block considerably the replication synthesis mediated by the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I. Strikingly, the polymerase incorporated incorrect nucleotides, dGMP and dAMP, opposite the lesion more preferentially than the correct nucleotide, dCMP.
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PMID:Stereospecific synthesis and characterization of oligodeoxyribonucleotides containing an N2-(1-carboxyethyl)-2'-deoxyguanosine. 1787 41

G[8-5m]T, a guanine-thymine intrastrand cross-link lesion where the C8 of guanine is covalently bonded to the neighboring 3'-thymine through its methyl carbon, was previously shown to form in an aqueous solution of duplex DNA upon exposure to gamma- or X-rays and in calf thymus DNA treated with Fenton reagents. Here, we employed LC-MS/MS and demonstrated for the first time that this lesion could be induced in a dose-dependent manner in human Hela-S3 cells upon exposure to gamma-rays. We further carried out in vitro replication studies on a substrate containing a site-specifically incorporated G[8-5m]T, and our results showed that the Klenow fragment of Escherichia coli DNA polymerase I stopped synthesis mostly after incorporating the correct nucleotide dAMP opposite the 3'-thymine moiety of the lesion. On the other hand, yeast Saccharomyces cerevisiae DNA polymerase eta (pol eta) was able to replicate past the cross-link lesion, but with markedly reduced efficiency in nucleotide incorporation opposite the 5'-guanine of the lesion. Steady-state kinetic analyses for nucleotide incorporation by yeast pol eta showed that the 5'-guanine portion of the lesion also decreased pronouncedly the fidelity of nucleotide incorporation; the insertion of dAMP and dGMP was favored over that of the correct nucleotide, dCMP. The above results support the conclusion that oxidative intrastrand cross-link lesions, if not repaired, can be cytotoxic and mutagenic.
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PMID:In vivo formation and in vitro replication of a guanine-thymine intrastrand cross-link lesion. 1792 46


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