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)

An octadecadeoxynucleotide, modified site-specifically with N-(deoxyguanosin-N2-yl)-2-(acetylamino)fluorene (dG-N2-AAF), was prepared by enzymatic synthesis from a comparably modified decamer and then used as a DNA template in primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I containing (exo+) or lacking (exo-) 3'-->5' exonuclease activity. Using exo- Klenow fragment and all four deoxynucleotide triphosphate (dNTPs), primer extension is blocked one base before and opposite dG-N2-AAF. A small fraction of the reaction product represents translesional synthesis, in which dAMP is incorporated opposite the lesion. Kinetic studies of base insertion and chain extension indicate that the frequency of dAMP insertion opposite dG-N2-AAF is higher than that of other deoxynucleotide monophosphates (dNMPs) and of N-(deoxyguanosin-8-yl)-2-(acetylamino)-fluorene (dG-C8-AAF); however, the rate of extension of dA.dG-N2-AAF from the 3' terminus was much lower than that of dA.dG-C8-AAF. We conclude that dG-N2-AAF is a miscoding lesion and capable of generating G-->T transversion mutations in cells.
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PMID:Nucleotide misincorporation on DNA templates containing N-(deoxyguanosin-N2-yl)-2-(acetylamino)fluorene. 811 21

An experimental system has been developed by which base substitutions and frameshift deletions can be quantitated in vitro, using two-phase 20% polyacrylamide gel electrophoresis. Oligodeoxynucleotides, modified site-specifically, were used as templates in primer extension reactions catalyzed by DNA polymerase alpha, polymerase beta, and the Klenow fragment of Escherichia coli DNA polymerase I, with and without 3'-->5' exonuclease activity. Lesions studied included 7,8-dihydro-8-oxodeoxyguanosine, 7,8-dihydro-8-oxodeoxyadenosine, O6-methyldeoxyguanosine, N-(deoxyguanosin-8-yl)-2-(acetylamino)fluorene, and N-(deoxyguanosin-8-yl)-2- aminofluorene. Products of translesional synthesis contained dC, dA, dG, or dT opposite the lesion or one- and two-base deletions and were separated using a two-phase polyacrylamide gel system. When a template containing 8-oxoguanine was used, dAMP and/or dCAMP was incorporated opposite the lesion, the relative amounts depending on the DNA polymerase used. In contrast, the nonmutagenic base, dTMP, was incorporated exclusively opposite 8-oxodA in reactions catalyzed by Klenow fragment and pol alpha. The improved resolution provided by the two-phase gel system revealed misincorporation of dGMP opposite 8-oxodA in reactions catalyzed by pol beta. dTMP and small amounts of dCMP were incorporated opposite the lesion on an O6MedG-modified template. The bulky adduct, dG-C8-AAF, principally produced deletions; in contrast, dG-C8-AF promoted incorporation of dCMP, a nonmutagenic base. This experimental system should prove useful for establishing the miscoding potential of defined lesions in DNA templates and in correlating this information with the mutagenic properties of DNA adducts observed in cells.
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PMID:Quantitation of base substitutions and deletions induced by chemical mutagens during DNA synthesis in vitro. 829 39

The mutagenic properties of 2-acetylaminofluorene-derived DNA adducts, including N-(deoxyguanosin-8-yl)-2-acetylaminofluorene, N-(deoxyguanosin-8-yl)-2-aminofluorene, N-(deoxyguanosin-N2-yl)-2-acetylaminofluorene, and several minor oxidation products have been explored, using site-specific techniques. Oligodeoxynucleotides containing a single AAF-derived DNA adduct were prepared by postsynthetic modification and used as templates in primer extension reactions catalyzed by bacterial and mammalian DNA polymerases. Base substitutions and deletions occurring during DNA synthesis were quantified. dG-C8-AAF promoted one- and two-base deletions and small amounts of incorporation of dCMP, dAMP, and/or dTMP opposite the lesion in reactions catalyzed by the 3'-->5' exonuclease-free Klenow fragment of DNA polymerase 1 (exo-) and polymerase alpha. dG-C8-AF did not miscode in reactions catalyzed by exo-; however, base misincorporation and deletions were observed in reactions with pol alpha. dG-N2-AAF promoted small amounts of dAMP incorporation in reactions catalyzed by exo-. The miscoding potential of minor oxidation products of dG-C8-AF was much higher than that of other adducts. Steady-state kinetics were used to measure frequencies of nucleotide insertion opposite the lesion and chain extension from the 3' terminus. Kinetic data were consistent with the results of primer extension studies. A mutation 'hot spot' was constructed and the influence of sequence context on the frequency of deletions generated by dG-C8-AAF was explored systematically in reactions catalyzed by exo-. Based on our results with aminofluorene DNA adducts, we propose a general mechanism for frameshift deletion mutagenesis. Site-specific methods also were used to establish the mutagenic potential of AAF-derived DNA adducts in mammalian cells. dG-C8-AAF and dG-C8-AF exhibited similar mutagenic specificities, predicting the occurrence of G-->T transversions and G-->A transitions in mammalian cells.
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PMID:Molecular mechanisms of mutagenesis by aromatic amines and amides. 920 40

Bovine pancreatic deoxyribonuclease I (DNaseI) has been used to footprint T7 (exo-) DNA polymerase bound to a model primer-template junction. The polymerase was blocked at a specific position either by the omission of dCTP from the reaction mix or by the presence of a N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dGuo-AAF) adduct. This lesion has been shown to be a severe block for several DNA polymerases, both in in vitro primer elongation experiments, and during the in vivo replication of AAF-monomodified single-stranded vectors. The footprints obtained with unmodified primer-template DNA define two protected domains separated by an inter-region that remains sensitive to DNaseI, and several hypersensitive sites located on both strands. Binding of the polymerase to AAF monomodified duplexes results in the same protection pattern as that obtained with the unmodified duplexes. However, the hypersensitive sites either disappear or are dramatically reduced. The results suggest that the AAF lesion alters the correct positioning of the duplex DNA within the polymerase cleft.
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PMID:A single N-2-acetylaminofluorene adduct alters the footprint of T7 (exo-) DNA polymerase bound to a model primer-template junction. 953 79

Oligonucleotides containing a single N-(deoxyguanosin-8-yl)acetylaminofluorene lesion (dGuo-C8-AAF) at each guanine residue of the sequence (5'-G1G2G3) have been used as templates for in vitro primer extension reactions by several DNA polymerases [Escherichia coli DNA polymerase III holoenzyme, its alpha subunit, DNA polymerase I Klenow fragment proficient (exo+) or deficient (exo-) in its 3' --> 5' exonuclease activity, and Sequenase]. The dGuo-C8-AAF lesion appears to be a strong block for all DNA polymerases: exo+ DNA polymerases stop one nucleotide before encountering the lesion, while partial incorporation opposite the lesion is observed only with enzymes devoid of the exonuclease activity. The efficiency of incorporation across from the adduct depends on both the DNA polymerase and the position of the lesion. When polymerase I Klenow fragment exo- is used, translesion synthesis (TLS) is observed with efficiencies varying according to the position of the adduct (G2 > G1 > G3). Sequencing of the TLS products shows that error-free TLS is observed only when the AAF lesion is bound to G1, while all TLS events occurring at G2- or G3-AAF adducts are mutagenic. The major mutational event is a G deletion (27, 76, and 55% of the events for G1, G2, and G3, respectively), while two-G deletions occur to a lesser extent (17-30%). These results are discussed in view of the slippage model developed for frameshift mutagenesis occurring during translesion synthesis at replication blocking lesions.
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PMID:Sequence context modulation of translesion synthesis at a single N-2-acetylaminofluorene adduct located within a mutation hot spot. 1002 91

The carcinogen N-acetyl-2-aminofluorene forms two major DNA adducts: the N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene adduct (dG-C8-AAF) and its deacetylated derivative, the N-(2'-deoxyguanosin-8-yl)-2-aminofluorene adduct (dG-C8-AF). It is well established that the AAF adduct is a very strong block for DNA synthesis in vitro while the AF adduct is more easily bypassed. In an effort to understand the molecular mechanism of this phenomenon, the structure of the complex of an exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment) bound to primer-templates containing either an AF or AAF adduct in or near the active site was probed by nuclease and protease digestion analyses. The results of these experiments suggest that positioning the AAF adduct in the polymerase active site strongly inhibits the conformational change that is required for the insertion of a nucleotide. Similar experiments with AF-modified primer-templates shows a much less pronounced effect. The inhibition of the conformational change by either adduct is not detected if they are positioned in the single-stranded part of the template just one nucleotide before the active site. These findings may explain the different abilities of these lesions to block DNA synthesis.
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PMID:Differential effects of N-acetyl-2-aminofluorene and N-2-aminofluorene adducts on the conformational change in the structure of DNA polymerase I (Klenow fragment). 1081 81

Lesion bypass is an important mechanism to overcome replication blockage by DNA damage. Translesion synthesis requires a DNA polymerase (Pol). Human Pol iota encoded by the RAD30B gene is a recently identified DNA polymerase that shares sequence similarity to Pol eta. To investigate whether human Pol iota plays a role in lesion bypass we examined the response of this polymerase to several types of DNA damage in vitro. Surprisingly, 8-oxoguanine significantly blocked human Pol iota. Nevertheless, translesion DNA synthesis opposite 8-oxoguanine was observed with increasing concentrations of purified human Pol iota, resulting in predominant C and less frequent A incorporation opposite the lesion. Opposite a template abasic site human Pol iota efficiently incorporated a G, less frequently a T and even less frequently an A. Opposite an AAF-adducted guanine, human Pol iota was able to incorporate predominantly a C. In both cases, however, further DNA synthesis was not observed. Purified human Pol iota responded to a template TT (6-4) photoproduct by inserting predominantly an A opposite the 3' T of the lesion before aborting DNA synthesis. In contrast, human Pol iota was largely unresponsive to a template TT cis-syn cyclobutane dimer. These results suggest a role for human Pol iota in DNA lesion bypass.
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PMID:Response of human DNA polymerase iota to DNA lesions. 1116 Sep 25

Human DNA polymerase kappa (pol kappa) has a sequence significantly homologous with that of Escherichia coli DNA polymerase IV (pol IV). We used a truncated form of human pol kappa (pol kappaDeltaC) and full-length pol IV to explore the miscoding properties of these enzymes. Oligodeoxynucleotides, modified site-specifically with N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-AF), were used as DNA templates in primer extension reactions that included all four dNTPs. Reactions catalyzed by pol kappaDeltaC were partially blocked one base prior to dG-AAF or dG-AF, and also opposite both lesions. At higher enzyme concentrations, a significant fraction of primer was extended. Analysis of the fully extended reaction product revealed incorporation of dTMP opposite dG-AAF, accompanied by much smaller amounts of dCMP, dAMP, and dGMP and some one- and two-base deletions. The product terminating 3' to the adduct site contained AMP misincorporated opposite dC. On templates containing dG-AF, dAMP, dTMP, and dCMP were incorporated opposite the lesion in approximately equal amounts, together with some one-base and two-base deletions. Steady-state kinetics analysis confirmed the results obtained from primer extension reactions catalyzed by pol kappa. In contract, primer extension reactions catalyzed by pol IV were blocked effectively by dG-AAF and dG-AF. At high concentrations of pol IV, full-length products were formed containing primarily one- or two-base deletions with dCMP, the correct base, incorporated opposite dG-AF. The miscoding properties of pol kappa observed in this study are consistent with mutational spectra observed when plasmid vectors containing dG-AAF or dG-AF are introduced into simian kidney cells [Shibutani, S., et al. (2001) Biochemistry 40, 3717-3722], supporting a model in which pol kappa plays a role in translesion synthesis past acetylaminofluorene-derived lesions in mammalian cells.
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PMID:Translesional synthesis past acetylaminofluorene-derived DNA adducts catalyzed by human DNA polymerase kappa and Escherichia coli DNA polymerase IV. 1173

Aromatic amines have been studied for more than a half-century as model carcinogens representing a class of chemicals that form bulky adducts to the C8 position of guanine in DNA. Among these guanine adducts, the N-(2'-deoxyguanosin-8-yl)-aminofluorene (G-AF) and N-2-(2'-deoxyguanosin-8-yl)-acetylaminofluorene (G-AAF) derivatives are the best studied. Although G-AF and G-AAF differ by only an acetyl group, they exert different effects on DNA replication by replicative and high-fidelity DNA polymerases. Translesion synthesis of G-AF is achieved with high-fidelity polymerases, whereas replication of G-AAF requires specialized bypass polymerases. Here we have presented structures of G-AF as it undergoes one round of accurate replication by a high-fidelity DNA polymerase. Nucleotide incorporation opposite G-AF is achieved in solution and in the crystal, revealing how the polymerase accommodates and replicates past G-AF, but not G-AAF. Like an unmodified guanine, G-AF adopts a conformation that allows it to form Watson-Crick hydrogen bonds with an opposing cytosine that results in protrusion of the bulky fluorene moiety into the major groove. Although incorporation opposite G-AF is observed, the C:G-AF base pair induces distortions to the polymerase active site that slow translesion synthesis.
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PMID:Observing translesion synthesis of an aromatic amine DNA adduct by a high-fidelity DNA polymerase. 1538 34

The carcinogen 2-acetylaminofluorene forms two major DNA adducts: N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and its deacetylated derivative, N-(2'-deoxyguanosin-8-yl)-2-aminofluorene (dG-AF). Although the dG-AAF and dG-AF adducts are distinguished only by the presence or absence of an acetyl group, they have profoundly different effects on DNA replication. dG-AAF poses a strong block to DNA synthesis and primarily induces frameshift mutations in bacteria, resulting in the loss of one or two nucleotides during replication past the lesion. dG-AF is less toxic and more easily bypassed by DNA polymerases, albeit with an increased frequency of misincorporation opposite the lesion, primarily resulting in G --> T transversions. We present three crystal structures of bacteriophage T7 DNA polymerase replication complexes, one with dG-AAF in the templating position and two others with dG-AF in the templating position. Our crystallographic data suggest why a dG-AAF adduct blocks replication more strongly than does a dG-AF adduct and provide a possible explanation for frameshift mutagenesis during replication bypass of a dG-AAF adduct. The dG-AAF nucleoside adopts a syn conformation that facilitates the intercalation of its fluorene ring into a hydrophobic pocket on the surface of the fingers subdomain and locks the fingers in an open, inactive conformation. In contrast, the dG-AF base at the templating position is not well defined by the electron density, consistent with weak binding to the polymerase and a possible interchange of this adduct between the syn and anti conformations.
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PMID:Crystal structures of 2-acetylaminofluorene and 2-aminofluorene in complex with T7 DNA polymerase reveal mechanisms of mutagenesis. 1552 77


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