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)

DNA polymerase beta (beta-pol), which is involved in base excision repair, was investigated for its role in protection of cells against various genotoxic agents and cytostatic drugs using beta-pol knockout mouse fibroblasts. We show that cells lacking beta-pol are highly sensitive to induction of apoptosis and chromosomal breakage by methylating agents, such as N-methyl-N'-nitro-N-nitrosoguanidine and methyl methanesulfonate and the cross-linking antineoplastic drugs mitomycin C and mafosfamide. The cross-sensitivity between the agents observed suggests that beta-pol is involved in repair not only of DNA methylation lesions but also of other kinds of DNA damage induced by various cytostatic drugs. Cells deficient in beta-pol were not hypersensitive to cisplatin, melphalan, benzo(a)pyrene diol epoxide, chloroethylnitrosourea, or UV light. Because both established and primary beta-pol knockout fibroblasts displayed the hypersensitive phenotype, which, moreover, was complemented by transfection with a beta-pol expression vector, the alkylating agent hypersensitivity can clearly be attributed to the beta-pol deficiency. The results demonstrate that beta-pol-driven base excision repair is highly important for protection of cells against cell killing due to apoptosis and induced chromosomal breakage and suggest that incompletely repaired DNA damage causes chromosomal changes and may act as a trigger of DNA damage-induced apoptosis.
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PMID:Cells deficient in DNA polymerase beta are hypersensitive to alkylating agent-induced apoptosis and chromosomal breakage. 1019 27

To enhance the specificity of polymerase photoaffinity labeling, a novel approach based on sensitized photomodification has been developed. A base-substituted analog of TTP containing a pyrene group (PyrdUTP) was synthesized and used as an active site-bound photosensitizer for photoaffinity modification of DNA polymerase beta (pol beta). 5'-[32P]-labeled primer was elongated in situ by pol beta with a photoreactive analog of TTP (FAB-4-dUTP). The pyrene sensitizer (PyrdUTP), excited by light (365-450 nm), can activate the photoreagent, cross-linking it to pol beta as a result of fluorescence resonance energy transfer. The initial rate of pol beta photomodification was shown to increase by a factor of ten. The selectivity of pol beta photosensitized modification was proved by adding human replication protein A.
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PMID:Sensitized photomodification of mammalian DNA polymerase beta. A new approach for highly selective affinity labeling of polymerases. 1021 27

The binding of the benzo[a]pyrene metabolite anti-BPDE (r7, t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene) to the N(2) group of 2'-deoxyguanosine residues (dG) is known to adversely affect the Michaelis-Menten primer extension kinetics catalyzed by DNA Pol I and other polymerases. In this work, the impact of site-specific, anti-BPDE-modified DNA template strands on the formation of Pol I (Klenow fragment, KF)/template-primer complexes has been investigated. The 23-mer template strand 5'-d(AAC GC-(1) T(-)(2) ACC ATC CGA ATT CGC CC), I (dG = (+)-trans- and (-)-trans-anti-BPDE-N(2)-dG), was annealed with primer strands 18, 19, or 20 bases long. Complex formation of these template-primer strands with KF(-) (exonuclease-free) at different enzyme concentrations was determined using polyacrylamide gel mobility shift assays in the absence of dNTPs. The lesion dG causes an increase in the dissociation constants, K(d), of the monomeric, 1:1 KF(-)/DNA template-primer complexes by factors of 10-15 when the 3'-end base of the primer strand is positioned either opposite dG, or opposite dC(-)(1) in I, and the shapes of the binding isotherms are sigmoidal. The sigmoidal shapes are attributed to the formation of dimeric 2:1 KF(-)/DNA template-primer complexes. In contrast, when the 3'-end of the primer strand extends only to dT(-)(2) in I, the K(d) of 1:1 complexes is increased by factors of only 2-3, the shapes of the binding isotherms are hyperbolic and nonsigmoidal and are similar to those observed with the unmodified control, and monomeric KF(-)/DNA complexes are dominant. The impact of bulky lesions on polymerase/DNA complex formation in polymerase-catalyzed primer extension reactions needs to be taken into account in interpreting the site-specific Michaelis-Menten kinetics of these reactions.
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PMID:Primer length dependence of binding of DNA polymerase I Klenow fragment to template-primer complexes containing site-specific bulky lesions. 1051 40

The process of carcinogenesis is initiated by mutagenesis, which often involves replication past damaged DNA. One question - what exactly is a DNA polymerase seeing when it incorrectly copies a damaged DNA base (e.g., inserting dATP opposite a dG adduct)? - has not been answered in any case. Herein, we reflect on this question, principally by considering the mutagenicity of one activated form of benzo[a]pyrene, (+)-anti-B[a]PDE, and its major adduct [+ta]-B[a]P-N(2)-dG. In previous work, [+ta]-B[a]P-N(2)-dG was shown to be capable of inducing>95% G-->T mutations in one sequence context (5'-TGC), and approximately 95% G-->A mutations in another (5'-AGA). This raises the question - how can a single chemical entity induce different mutations depending upon DNA sequence context? Our current working hypothesis is that adduct conformational complexity causes adduct mutational complexity, where DNA sequence context can affect the former, thereby influencing the latter. Evidence supporting this hypothesis was discussed recently (Seo et al., Mutation Res. [in press]). Assuming this hypothesis is correct (at least in some cases), one goal is to consider what these mutagenic conformations might be. Based on molecular modeling studies, 16 possible conformations for [+ta]-B[a]P-N(2)-dG are proposed. A correlation between molecular modeling and mutagenesis work suggests a hypothesis (Hypothesis 3): a base displaced conformation with the dG moiety of the adduct in the major vs. minor groove gives G-->T vs. G-->A mutations, respectively. (Hypothesis 4, which is a generalized version of Hypothesis 3, is also proposed, and can potentially rationalize aspects of both [+ta]-B[a]P-N(2)-dG and AP-site mutagenesis, as well as the so-called "A-rule".) Finally, there is a discussion of how conformational complexity might explain some unusual mutagenesis results that suggest [+ta]-B[a]P-N(2)-dG can become trapped in different conformations, and why we think it makes sense to interpret adduct mutagenesis results by modeling ds-DNA (at least in some cases), even though the mutagenic event must occur at a ss/ds-DNA junction in the presence of a DNA polymerase.
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PMID:Toward an understanding of the role of DNA adduct conformation in defining mutagenic mechanism based on studies of the major adduct (formed at N(2)-dG) of the potent environmental carcinogen, benzo[a]pyrene. 1083 33

The presence of benzo[a]pyrene diol epoxide (B[a]PDE) adducts in DNA is known to interfere with DNA replication. Kinetic studies of nucleotide insertion by exonuclease-deficient E. coli DNA polymerase I (Klenow fragment) across from either the (+)-trans- or the (+)-cis-B[a]P-N(2)-dG adduct in the 5'-CGT-3' sequence context indicated that the rate of nucleotide incorporation followed the order: dAMP > dGMP > dTMP > dCMP, which did not correlate with the mutational spectrum observed for these adducts in this sequence in E. coli (mostly G-->A transitions). Interestingly, a kinetic analysis of extension past the adduct showed that, unlike other sequences studied, the primer-template was extended best when dT was positioned at the 3'-terminus of the primer across from either a (+)-trans- or a (+)-cis-B[a]P-N(2)-dG adduct. In contrast, when the (+)-trans-B[a]P-N(2)-dG adduct was positioned in the 5'-TGC-3' sequence context, which gives predominantly G-->T mutations in E. coli, extension was detectable only when dA was positioned across from the adduct. These data provide the first in vitro evidence that may explain why G-->A transitions, rather than the G-->T transversions found in other sequences, are preferred in the 5'-CGT-3' sequence in vivo.
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PMID:In vitro replication of primer-templates containing benzo[a]pyrene adducts by exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment): effect of sequence context on lesion bypass. 1095 33

Error-free lesion bypass and error-prone lesion bypass are important cellular responses to DNA damage during replication, both of which require a DNA polymerase (Pol). To identify lesion bypass DNA polymerases, we have purified human Polkappa encoded by the DINB1 gene and examined its response to damaged DNA templates. Here, we show that human Polkappa is a novel lesion bypass polymerase in vitro. Purified human Polkappa efficiently bypassed a template 8-oxoguanine, incorporating mainly A and less frequently C opposite the lesion. Human Polkappa most frequently incorporated A opposite a template abasic site. Efficient further extension required T as the next template base, and was mediated mainly by a one-nucleotide deletion mechanism. Human Polkappa was able to bypass an acetylaminofluorene-modified G in DNA, incorporating either C or T, and less efficiently A opposite the lesion. Furthermore, human Polkappa effectively bypassed a template (-)-trans-anti-benzo[a]pyrene-N:(2)-dG lesion in an error-free manner by incorporating a C opposite the bulky adduct. In contrast, human Polkappa was unable to bypass a template TT dimer or a TT (6-4) photoproduct, two of the major UV lesions. These results suggest that Polkappa plays an important role in both error-free and error-prone lesion bypass in humans.
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PMID:Error-free and error-prone lesion bypass by human DNA polymerase kappa in vitro. 1105 10

We recently proposed a mechanism for why dAMP is primarily inserted opposite both T's of photoproducts of TT sites by T7 DNA polymerase [Smith, C. A., Baeten, J., and Taylor, J.-S. (1998) J. Biol. Chem., 273, 21933-21940] that was based on analysis of a recent crystal structure of a complex of this enzyme with a template, a primer, and a dideoxynucleotide. We proposed that indiscriminate insertion of dAMP opposite the 3'-T of each photoproducts takes place via a transient abasic site-like intermediate, with the photoproduct outside the active site, whereas insertion of dAMP opposite the 5'-T takes place with the photoproduct inside the active site. To obtain further support for this mechanism, we have investigated the selectivity of dNMP and pyrene nucleotide (dPMP) insertion opposite each T of the cis,syn, trans,syn-I, trans,syn-II, (6-4), and Dewar photoproducts of TT and opposite a tetrahydrofuran abasic site analogue by the exonuclease-deficient T7 DNA polymerase, Sequenase Version 2.0. Selectivity was determined by a direct competition assay that makes use of a stacked gel to resolve the various extension products. Pyrene nucleotide was chosen for investigation because it has been previously shown to be selectively inserted opposite abasic sites and was therefore expected to probe whether the photoproducts were inside the active site during a particular insertion step. In accord with the proposed mechanism, dPMP was inserted in preference to dAMP opposite the 3'-T of all the photoproducts with the exception of the trans,syn-I product, whereas dAMP was inserted in preference to dPMP opposite the 5'-T of all the photoproducts. In addition to supporting the proposed mechanism, these results suggest that pyrene nucleotide may be a useful probe for investigating the mechanism of DNA damage bypass by polymerases and for characterizing their active sites.
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PMID:Pyrene nucleotide as a mechanistic probe: evidence for a transient abasic site-like intermediate in the bypass of dipyrimidine photoproducts by T7 DNA polymerase. 1108 16

DNA replication fidelity is dictated by DNA polymerase enzymes and associated proteins. When the template DNA is damaged by a carcinogen, the fidelity of DNA replication is sometimes compromized, allowing mispaired bases to persist and be incorporated into the DNA, resulting in a mutation. A key question in chemical carcinogenesis by metabolically activated polycyclic aromatic hydrocarbons (PAHs) is the nature of the interactions between the carcinogen-damaged DNA and the replicating polymerase protein that permits the mutagenic misincorporation to occur. PAHs are environmental carcinogens that, upon metabolic activation, can react with DNA to form bulky covalently linked combination molecules known as carcinogen-DNA adducts. Benzo[a]pyrene (BP) is a common PAH found in a wide range of material ingested by humans, including cigarette smoke, car exhaust, broiled meats and fish, and as a contaminant in other foods. BP is metabolically activated into several highly reactive intermediates, including the highly tumorigenic (+)-anti-benzo[a]pyrene diol epoxide (BPDE). The primary product of the reaction of (+)-anti-BPDE with DNA, the (+)-trans-anti-benzo[a]pyrene diol epoxide-N(2)-dG ((+)-ta-[BP]G) adduct, is the most mutagenic BP adduct in mammalian systems and primarily causes G-to-T transversion mutations, resulting from the mismatch of adenine with BP-damaged guanine during replication. In order to elucidate the structural characteristics and interactions between the DNA polymerase and carcinogen-damaged DNA that allow a misincorporation opposite a DNA lesion, we have modeled a (+)-ta-[BP]G adduct at a primer-template junction within the replicative phage T7 DNA polymerase containing an incoming dATP, the nucleotide most commonly mismatched with the (+)-ta-[BP]G adduct during replication. A one nanosecond molecular dynamics simulation, using AMBER 5.0, has been carried out, and the resultant trajectory analyzed. The modeling and simulation have revealed that a (+)-ta-[BP]G:A mismatch can be accommodated stably in the active site so that the fidelity mechanisms of the polymerase are evaded and the polymerase accepts the incoming mutagenic base. In this structure, the modified guanine base is in the syn conformation, with the BP moiety positioned in the major groove, without interfering with the normal protein-DNA interactions required for faithful polymerase function. This structure is stabilized by a hydrogen bond between the modified guanine base and dATP partner, hydrophobic interactions between the BP moiety and the polymerase, a hydrogen bond between the modified guanine base and the polymerase, and several hydrogen bonds between the BP moiety and polymerase side-chains. Moreover, the G:A mismatch in this system closely resembles the size and shape of a normal Watson-Crick pair. These features reveal how the polymerase proofreading machinery may be evaded in the presence of a mutagenic carcinogen-damaged DNA, so that a mismatch can be accommodated readily, allowing bypass of the adduct by the replicative T7 DNA polymerase.
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PMID:Evading the proofreading machinery of a replicative DNA polymerase: induction of a mutation by an environmental carcinogen. 1137 Nov 69

The effects of base sequence, specifically different pyrimidines flanking a bulky DNA adduct, on translesional synthesis in vitro catalyzed by the Klenow fragment of Escherichia coli Pol I (exo(-)) was investigated. The bulky lesion was derived from the binding of a benzo[a]pyrene diol epoxide isomer [(+)-anti-BPDE] to N(2)-guanine (G*). Four different 43-base long oligonucleotide templates were constructed with G* at a site 19 bases from the 5'-end. All bases were identical, except for the pyrimidines, X or Y, flanking G* (sequence context 5'-.XGY., with X, Y = C and/or T). In all cases, the adduct G* slows primer extension beyond G* more than it slows the insertion of a dNTP opposite G* (A and G were predominantly inserted opposite G, with A > G). Depending on X or Y, full lesion bypass differed by factors of approximately 1.5-5 ( approximately 0.6-3.0% bypass efficiencies). A downstream T flanking G on the 5'-side instead of C favors full lesion bypass, while an upstream C flanking G* is more favorable than a T. Various deletion products resulting from misaligned template-primer intermediates are particularly dominant ( approximately 5.0-6.0% efficiencies) with an upstream flanking C, while a 3'-flanking T lowers the levels of deletion products ( approximately 0.5-2.5% efficiencies). The kinetics of (1) single dNTP insertion opposite G* and (2) extension of the primer beyond G* by a single dNTP, or in the presence of all four dNTPs, with different 3'-terminal primer bases (Z) opposite G* were investigated. Unusually efficient primer extension efficiencies beyond the adduct (approaching approximately 90%) was found with Z = T in the case of sequences with 3'-flanking upstream C rather than T. These effects are traced to misaligned slipped frameshift intermediates arising from the pairing of pairs of downstream template base sequences (up to 4-6 bases from G*) with the 3'-terminal primer base and its 5'-flanking base. The latter depend on the base Y and on the base preferentially inserted opposite the adduct. Thus, downstream template sequences as well as the bases flanking G* influence DNA translesion synthesis.
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PMID:Base sequence dependence of in vitro translesional DNA replication past a bulky lesion catalyzed by the exo- Klenow fragment of Pol I. 1138 Feb 61

To increase the efficiency of photoaffinity labeling of DNA polymerases, a binary system of photoaffinity reagents was applied. Photoreactive radioactive primers were synthesized by DNA polymerases beta (pol beta) or DNA polymerase from Thermus thermophilus (pol Tte) using a template-primer duplex in the presence of a dTTP analogue containing 4-azidotetrafluorobenzoyl group linked via spacers of varying length to 5-position of uridine ring- 5-[N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (FAB-4-dUTP) or 5-[N-[[(2,3,5,6-tetrafluoro-4-azidobenzoyl)-butanoyl]-amino]-trans-3-aminopropenyl-1]-2'-deoxyuridine-5'-triphosphate (FAB-9-dUTP). The reaction mixtures were UV irradiated (lambda = 365-450 nm) in the absence or presence of a dTTP analog, containing a pyrene moiety-5-[N-(4-(1-pyrenyl)-butylcarbonyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (Pyr- 8-dUTP) or 5-[N-(4-(1-pyrenyl)-ethylcarbonyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (Pyr-6-dUTP). The most efficient crosslinking of both DNA polymerases was observed in the case of photoreactive DNA primer, carrying the FAB-4-dUMP moiety at the 3'-end, and Pyr-6-dUTP as a sensitizer. The binary system of photoaffinity reagents allows increasing photoaffinity labeling of the both DNA polymerases in comparison to the primer crosslinking without photosensitizer.
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PMID:A binary system of photoreagents for high-efficiency labeling of DNA polymerases. 1155 61


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