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)

Oxidative stress enhances lipid peroxidation (LPO) implicated in the promotion and progression of carcinogenesis. One of the major LPO products is trans-4-hydroxy-2-nonenal (HNE), which was shown to react with guanosine and under peroxidizing conditions also with adenosine. We show here that all four DNA bases are targets for HNE, although displaying different reactivity: dG > dC > dA approximately equal to dT. HPLC and mass spectrometry analyses of HNE reactions with deoxynucleosides showed in each case the formation of several products, with mass peaks corresponding to HNE-dN adducts at a 1:1 and also 2:1 and 3:1 ratios. In the dA, dC and dG reactions, mass peaks corresponding to heptyl-substituted etheno-adducts were also detected, indicating HNE oxidation to its epoxide by air oxygen. In DNA pretreated with HNE, DNA synthesis by T7 DNA polymerase was stopped in a sequence-dependent manner at G > or = C > A and T sites. HNE increased the mutation rates in the lac Z gene of M13 phage transfected into wild type Escherichia coli. The most frequent event was the recombination between lacZ gene sequences in M13 and the E. coli F' factor DNA. Base substitutions and frameshifts were also observed in approximately similar numbers. Over 50% of base substitutions were the C-->T transitions, followed by the G-->C and A-->C transversions. In the E. coli recA strain recombination was not observed, although one mutational G-->T hot-spot appeared within the DNA fragment undergoing recombination in the wild type E. coli. We conclude that long chain HNE adducts to DNA bases arrest DNA synthesis and cause recombination, base substitutions and frameshift mutations in ssDNA.
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PMID:Long-chain adducts of trans-4-hydroxy-2-nonenal to DNA bases cause recombination, base substitutions and frameshift mutations in M13 phage. 1513 39

Interactions between the minor groove of the DNA and DNA polymerases appear to play a major role in the catalysis and fidelity of DNA replication. In particular, Arg668 of Escherichia coli DNA polymerase I (Klenow fragment) makes a critical contact with the N-3-position of guanine at the primer terminus. We investigated the interaction between Arg668 and the ring oxygen of the incoming deoxynucleotide triphosphate (dNTP) using a combination of site-specific mutagenesis of the protein and atomic substitution of the DNA and dNTP. Hydrogen bonds from Arg668 were probed with the site-specific mutant R668A. Hydrogen bonds from the DNA were probed with oligodeoxynucleotides containing either guanine or 3-deazaguanine (3DG) at the primer terminus. Hydrogen bonds from the incoming dNTP were probed with (1 'R,3 'R,4 'R)-1-[3-hydroxy-4-(triphosphorylmethyl)cyclopent-1-yl]uracil (dcUTP), an analog of dUTP in which the ring oxygen of the deoxyribose moiety was replaced by a methylene group. We found that the pre-steady-state parameter kpol was decreased 1,600 to 2,000-fold with each of the single substitutions. When the substitutions were combined, there was no additional decrease (R668A and 3DG), a 5-fold decrease (3DG and dcUTP), and a 50-fold decrease (R668A and dcUTP) in kpol. These results are consistent with a hydrogen-bonding fork from Arg668 to the primer terminus and incoming dNTP. These interactions may play an important role in fidelity as well as catalysis of DNA replication.
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PMID:Escherichia coli DNA polymerase I (Klenow fragment) uses a hydrogen-bonding fork from Arg668 to the primer terminus and incoming deoxynucleotide triphosphate to catalyze DNA replication. 1521 Jul 7

Oxidative stress plays an important role in tissue damage caused by hypoglycemia and diabetes, which may be the result of deterioration in glucose homeostasis caused by these metabolic disorders. The present study examined the effects of insulin-induced hypoglycemia and streptozotocin induced diabetes on mitochondrial lipid peroxidation and antioxidant enzymes from different brain regions, namely, cerebral hemispheres, cerebellum, brain stem and diencephalon. In situ localization of DNA single strand breaks (SSBs) were also studied by DNA polymerase-I mediated biotin dATP labeled nick translation method after inducing hypoglycemia and diabetes. Significant decrease in mitochondrial catalase, manganese superoxide-dismutase (Mn-SOD) and reduced glutathione (GSH) content and increase in the lipid peroxidation (LPx) and glutathione peroxidase (GPx) activity was observed under these metabolic stress conditions with more pronounced effects in hypoglycemic group. We conclude that during severe energy deprivation following hypoglycemia and diabetes, mitochondrial free radicals scavenger system is down regulated, which leads to reactive oxygen species (ROS) generation. High levels of ROS in turn activate the processes leading to DNA damage. DNA SSBs, which indicates nuclear disintegration is an important feature of neuronal cell death.
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PMID:Impact of hypoglycemia and diabetes on CNS: correlation of mitochondrial oxidative stress with DNA damage. 1522 97

Oxanine (Oxa) is a deaminated base lesion derived from guanine in which the N(1)-nitrogen is substituted by oxygen. This work reports the mutagenicity of oxanine as well as oxanine DNA glycosylase (ODG) activities in mammalian systems. Using human DNA polymerase beta, deoxyoxanosine triphosphate is only incorporated opposite cytosine (Cyt). When an oxanine base is in a DNA template, Cyt is efficiently incorporated opposite the template oxanine; however, adenine and thymine are also incorporated opposite Oxa with an efficiency approximately 80% of a Cyt/Oxa (C/O) base pair. Guanine is incorporated opposite Oxa with the least efficiency, 16% compared with cytosine. ODG activity was detected in several mammalian cell extracts. Among the known human DNA glycosylases tested, human alkyladenine glycosylase (AAG) shows ODG activity, whereas hOGG1, hNEIL1, or hNEIL2 did not. ODG activity was detected in spleen cell extracts of wild type age-matched mice, but little activity was observed in that of Aag knock-out mice, confirming that the ODG activity is intrinsic to AAG. Human AAG can excise Oxa from all four Oxa-containing double-stranded base pairs, Cyt/Oxa, Thy/Oxa, Ade/Oxa, and Gua/Oxa, with no preference to base pairing. Surprisingly, AAG can remove Oxa from single-stranded Oxa-containing DNA as well. Indeed, AAG can also remove 1,N(6)-ethenoadenine from single-stranded DNA. This study extends the deaminated base glycosylase activities of AAG to oxanine; thus, AAG is a mammalian enzyme that can act on all three purine deamination bases, hypoxanthine, xanthine, and oxanine.
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PMID:Oxanine DNA glycosylase activity from Mammalian alkyladenine glycosylase. 1524 9

Accurate DNA replication involves polymerases with high nucleotide selectivity and proofreading activity. We show here why both fidelity mechanisms fail when normally accurate T7 DNA polymerase bypasses the common oxidative lesion 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8oG). The crystal structure of the polymerase with 8oG templating dC insertion shows that the O8 oxygen is tolerated by strong kinking of the DNA template. A model of a corresponding structure with dATP predicts steric and electrostatic clashes that would reduce but not eliminate insertion of dA. The structure of a postinsertional complex shows 8oG(syn).dA (anti) in a Hoogsteen-like base pair at the 3' terminus, and polymerase interactions with the minor groove surface of the mismatch that mimic those with undamaged, matched base pairs. This explains why translesion synthesis is permitted without proofreading of an 8oG.dA mismatch, thus providing insight into the high mutagenic potential of 8oG.
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PMID:Structural basis for the dual coding potential of 8-oxoguanosine by a high-fidelity DNA polymerase. 1529 82

Aerobic respiration generates reactive oxygen species that can damage guanine residues and lead to the production of 8-oxoguanine (8oxoG), the major mutagenic oxidative lesion in the genome. Oxidative damage is implicated in ageing and cancer, and its prevalence presents a constant challenge to DNA polymerases that ensure accurate transmission of genomic information. When these polymerases encounter 8oxoG, they frequently catalyse misincorporation of adenine in preference to accurate incorporation of cytosine. This results in the propagation of G to T transversions, which are commonly observed somatic mutations associated with human cancers. Here, we present sequential snapshots of a high-fidelity DNA polymerase during both accurate and mutagenic replication of 8oxoG. Comparison of these crystal structures reveals that 8oxoG induces an inversion of the mismatch recognition mechanisms that normally proofread DNA, such that the 8oxoG.adenine mismatch mimics a cognate base pair whereas the 8oxoG.cytosine base pair behaves as a mismatch. These studies reveal a fundamental mechanism of error-prone replication and show how 8oxoG, and DNA lesions in general, can form mismatches that evade polymerase error-detection mechanisms, potentially leading to the stable incorporation of lethal mutations.
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PMID:Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase. 1532 58

For the last ten years, antiretroviral therapy (ARV) has improved the prognosis in HIV-1 infection and showed a better control of the viral excretion by reducing viral shedding in semen. However, nucleoside analogues reverse transcriptase inhibitors (NRTI) therapy reported important adverse effects. Most of these side effects observed seem to be linked with a common mechanism: mitochondrial activity alteration. Since the introduction of protocols for HIV-1 serodiscordant couples, with male infected partners under NRTI therapy, many results in the literature such as: semen characteristics and pregnancies, drew the attention of research teams. Many studies have suggested that NRTI has an affect on semen parameters, but proposed mechanisms of these effects have rarely been discussed. NRTI have a great affinity for the reverse transcriptase of HIV-1. Because many NRTI are not only inhibitors of reverse transcriptase but also inhibitors of the DNA polymerase beta and gamma, several toxic effects can be considered. Nevertheless, this specificity is not absolute and "accidental" incorporations of NRTI can occur on genomic sperm DNA. Only one study on genomic sperm DNA with patients under NRTI therapy was published without concluding results. Recently, studies have suggested that NRTI exposure could induce an alteration on mitochondrial energy-generating ability of spermatozoa. NRTI are known to induce an increase in the generation of reactive oxygen species, which results in the degradation of mitochondrial transmembrane potential (Deltapsim). This loss of Deltapsim can tend to release some specific apoptosis factors, such as cytochrome c, that initiates programmed cell death. Sperm DNA fragmentation, associated to apoptosis, was reported as a possible cause of recurrent pregnancy loss. If the incorporation of NRTI was reported in genomic DNA of somatic cells, the absence of data on the genomic sperm DNA justifies further studies concerning the effects of paternal exposure to NRTI on the genomic material of the male gamete, in particular because of its implication in the zygote development after fertilization.
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PMID:[Impact of reverse transcriptase inhibitors on sperm mitochondrial and genomic DNA in assisted reproduction techniques]. 1550 Nov 59

A series of six oligonucleotides with dihydrodiol epoxide metabolites of the polycyclic aromatic hydrocarbons (PAHs) benz[a]anthracene and benzo[a]pyrene attached to adenine N6 and guanine N2 atoms were prepared and studied with the processive bacteriophage DNA polymerase T7, exonuclease- (T7-). HIV-1 reverse transcriptase was much less efficient in polymerization than T7-. Benz[a]anthracene and benzo[a]pyrene adducts strongly blocked incorporation of dTTP and dCTP opposite the A and G derivatives, respectively. dATP was preferentially incorporated in all cases. Steady state kinetic analysis indicated that the low catalytic efficiency with adducted DNA was due to both increased K(m) and lowered k(cat) values. Some differences due to PAH stereochemistry were observed. Fluorescence estimates of K(d) and presteady state kinetic measurements of k(off) showed no major decrease in the affinity of T7- with damaged DNA substrates or with dNTPs. Presteady state kinetics showed a lack of the normal burst kinetics for dNTP incorporation with all PAH-DNA derivatives. These results indicate that the rate-limiting step is at or before the step of phosphodiester bond formation; release of the oligonucleotide is no longer the slowest step. Thio elemental effects (substitution of alpha-oxygen with sulfur) were relatively small, in contrast to previous work with T7- and 8-oxo-7,8-dihydroguanine. The effect of these bulky PAH adducts is either to attenuate rates of conformational changes or to introduce an additional conformation problem but not to alter the inherent affinity of the polymerase for DNA or dNTPs.
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PMID:Kinetics of nucleotide incorporation opposite polycyclic aromatic hydrocarbon-DNA adducts by processive bacteriophage T7 DNA polymerase. 1572 Jan 47

Base excision repair (BER) averts the cytotoxic and mutagenic effects of most endogenously produced DNA damage, including lesions that arise spontaneously due to the intrinsic instability of DNA or modifications that are formed from reactions with intracellular chemicals, such as reactive oxygen species and alkylating agents. Defects in the BER process have been associated with cancer susceptibility and neurodegenerative disorders. In its most simplistic form, BER can be fully reconstituted with a minimum of four human proteins and is completed in just five sequential steps: (i) excision of an inappropriate base by a DNA glycosylase (e.g., uracil DNA glycosylase); (ii) incision of the DNA backbone immediately adjacent to the resulting abasic site by apurinic/apyrimidimic endonuclease 1; (iii) removal of the 5'-abasic terminal fragment, and (iv) repair synthesis to fill the gap by DNA polymerase beta; and (v) ligation to seal the remaining nick by DNA ligase 1 or a complex of DNA ligase 3 and X-ray repair cross-complementing 1. However, BER can involve the participation of other proteins as well, such as alternative DNA polymerases or one of several nonessential "auxiliary" factors. In addition, BER operates most efficiently when specific protein-protein coordination occurs. Furthermore, several BER protein activities have been shown to be regulated by posttranslational modification, and some of the physical protein interactions link BER to other DNA transaction pathways. In this review, we summarize the current state of the emerging complexities of mammalian BER, focusing on the growing number of reported protein-protein interactions and posttranslational modifications.
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PMID:Protein-protein interactions and posttranslational modifications in mammalian base excision repair. 1580 10

The hydrogen bonding interactions between the Klenow fragment of Escherichia coli DNA polymerase I with the proofreading exonuclease inactivated (KF(-)) and the minor groove of DNA were examined with modified oligodeoxynucleotides in which 3-deazaguanine (3DG) replaced guanine. This substitution would prevent a hydrogen bond from forming between the polymerase and that one site on the DNA. If the hydrogen bonding interaction were important, then we should observe a decrease in the rate of reaction. The steady-state and pre-steady-state kinetics of DNA replication were measured with 10 different oligodeoxynucleotide duplexes in which 3DG was placed at different positions. The largest decrease in the rate of replication was observed when 3DG replaced guanine at the 3'-terminus of the primer. The effect of this substitution on mispair extension and formation was then probed. The G to 3DG substitution at the primer terminus decreased the k(pol) for the extension past G/C, G/A, and G/G base pairs but not the G/T base pair. The G to 3DG substitution at the primer terminus also decreased the formation of correct base pairs as well as incorrect base pairs. However, in all but two mispairs, the effect on correct base pairs was much greater than that of mispairs. These results indicate that the hydrogen bond between Arg668 and the minor groove of the primer terminus is important in the fidelity of both formation and extension of mispairs. These experiments support a mechanism in which Arg668 forms a hydrogen bonding fork between the minor groove of the primer terminus and the ring oxygen of the deoxyribose moiety of the incoming dNTP to align the 3'-hydroxyl group with the alpha-phosphate of the dNTP. This is one mechanism by which the polymerase can use the geometry of the base pairs to modulate the rate of formation and extension of mispairs.
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PMID:Fidelity of mispair formation and mispair extension is dependent on the interaction between the minor groove of the primer terminus and Arg668 of DNA polymerase I of Escherichia coli. 1582 23


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