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 design, synthesis and biological evaluation of novel seco-iso-cyclopropylfurano[2,3-e]indoline (seco-iso-CFI) and the seco-cyclopropyltetrahydrofurano[2,3-f]quinoline (seco-CFQ) analogues of the duocarmycins are described. These novel analogues (4-7) were designed on the premise that the lone pair of electrons on the furano-oxygen atom could enter into conjugation with the isocyclopropylfurano[e]indolone (iso-CFI) alkylating moiety, formed from the loss of HCl in compounds 4-7. The seco-iso-CFI DNA alkylating pharmacophore was synthesized through a well precedented approach of 5-exo-trig aryl radical cyclization with a vinyl chloride. In our studies, in addition to the formation of the seco-iso-CFI product, an equal amount of an unexpected seco-CFQ product was also generated during the radical cyclization reaction. Like CC-1065 and adozelesin, using Taq DNA polymerase stop and thermal cleavage assays, the seco-iso-CFI compounds (4 and 6) and the seco-CFQ compounds (5 and 7) were shown to preferentially alkylate the adenine-N3 position within the minor groove of long stretches of A residues. A MM2 energy optimized molecular model of a 1:1 complex of compound 6 with DNA reveals that the iso-CFI compound fits snugly within the minor groove. Using a MTT based experiment, the cytotoxicity of compounds 4-7 were determined against the growth of murine leukemia (L1210), mastocytoma (P815) and melanoma (B16) cell lines. The concentrations of compounds required to inhibit the growth of these tumor cells by 50% is in the range of 10(-8)M. These compounds were also tested against a panel of human cancer cells by the National Cancer Institute, demonstrating that the compounds exhibited a high level of activity against selected solid tumors. At a concentration of 0.0084 microM (based on the IC(50) of compound 17 (seco-CBI-TMI) against the growth L1210 cells), while compounds 4 and 17 were toxic against murine bone marrow cells as judged by a colony forming study of freshly isolated murine progenitor hematopoeitic cells, compound 5, a seco-CFQ compound, was significantly less toxic. Flow cytometric analysis of P815 cells that had been incubated for 24h with compounds 4 and 5 at their cytotoxic IC(50) concentrations indicated the induction of apoptosis in a large percentage of cells, thereby suggesting that this might be the mechanism by which the iso-CFI compounds kill cells.
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PMID:Novel furano analogues of duocarmycin C1 and C2: design, synthesis, and biological evaluation of seco-iso-cyclopropylfurano[2,3-e]indoline (seco-iso-CFI) and seco-cyclopropyltetrahydrofurano[2,3-f]quinoline (seco-CFQ) analogues. 1211 Mar 16

We had shown previously that DNA polymerase beta (beta-pol) null mouse fibroblasts, deficient in base excision repair (BER), are hypersensitive to monofunctional methylating agents but not to hydrogen peroxide (H2O2). This is surprising because beta-pol is thought to be involved in BER of oxidative as well as methylated DNA damage. We confirm these findings here in early-passage cells. However, with time in culture, beta-pol null cells become hypersensitive to H2O2 and other reactive oxygen species-generating agents. Analysis of in vitro BER reveals a strong deficiency in single-nucleotide BER of 8-oxoguanine (8-oxoG) by both early- and late-passage beta-pol null cell extracts. Therefore, in early-passage wild-type and beta-pol null cells, the capacity for single-nucleotide BER of 8-oxoG does not correlate with cellular sensitivity to H2O2. Expression of beta-pol protein in the late-passage null cells almost completely reverses the H2O2-hypersensitivity phenotype. Methoxyamine (MX) treatment sensitizes late-passage wild-type cells to H2O2 as expected for beta-pol-mediated single-nucleotide BER; however in beta-pol null cells, MX has no effect. The data indicate a role(s) of beta-pol-dependent repair in protection against the cytotoxicity of oxidative DNA damage in wild-type cells.
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PMID:Involvement of DNA polymerase beta in protection against the cytotoxicity of oxidative DNA damage. 1250 50

Oxidative damage to DNA generates 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). During DNA replication and repair synthesis, 8-oxodG can pair with cytosine or adenine. The ability to accurately replicate through this lesion depends on the DNA polymerase. We report the first structure of a polymerase with a promutagenic DNA lesion, 8-oxodG, in the confines of its active site. The modified guanine residue is in an anti conformation and forms Watson-Crick hydrogen bonds with an incoming dCTP. To accommodate the oxygen at C8, the 5'-phosphate backbone of the templating nucleotide flips 180 degrees. Thus, the flexibility of the template sugar-phosphate backbone near the polymerase active site is one parameter that influences the anti-syn equilibrium of 8-oxodG. Our results provide insights into the mechanisms employed by polymerases to select the complementary dNTP.
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PMID:Structure of DNA polymerase beta with the mutagenic DNA lesion 8-oxodeoxyguanine reveals structural insights into its coding potential. 1251 31

Thioredoxins are small ubiquitous proteins that display different functions mainly via redox-mediated processes. The facultatively photosynthetic bacterium Rhodobacter capsulatus harbours at least two genes for thioredoxin 1 and 2, trxA and trxC. It is demonstrated that thioredoxin 2 of R. capsulatus can partially replace the thioredoxin 1 function as a hydrogen donor for methionine sulfoxide reductase but cannot replace thioredoxin 1 as a subunit of phage T7 DNA polymerase. By inactivating the trxC gene in R. capsulatus, it is shown that thioredoxin 2 is involved in resistance against oxidative stress. As thioredoxin 1 of Rhodobacter sphaeroides, R. capsulatus thioredoxin 2 affects the oxygen-dependent expression of photosynthesis genes, albeit in an opposite way. The trxC mutant of R. capsulatus shows a stronger increase in photosynthesis gene expression after a decrease in oxygen tension than the isogenic wild-type strain. The expression of the trxC gene is downregulated by oxygen.
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PMID:Thioredoxin 2 is involved in oxidative stress defence and redox-dependent expression of photosynthesis genes in Rhodobacter capsulatus. 1262 4

6-Anilinopyrazolo[3,4-d]pyrimidin-4-ones are novel dGTP analogues that inhibit the replication-specific enzyme DNA polymerase III (DNA pol III) of Staphlococcus aureus and other Gram-positive (Gr+) bacteria. To enhance the potential of these inhibitors as antimicrobial agents, a structure-activity relationship was developed involving substitutions at the 2, 4, and pyrazolo NH positions. All of the new inhibitors were tested for their ability to inhibit S. aureus DNA pol III and the growth of several other Gr+ bacteria in culture. 2-Anilino groups with small hydrophobic groups in the meta or para position enhanced both antipolymerase and antimicrobial activity. 2-Benzyl-substituted inhibitors were substantially less active. Substitution in the 4-position by oxygen gave the optimal activity, whereas substitution at the pyrazolo NH was not tolerated. These pyrazolo[3,4-d]pyrimidine derivatives represent a novel class of antimicrobials with promising activities against Gr+ bacteria.
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PMID:Novel pyrazolo[3,4-d]pyrimidine-based inhibitors of Staphlococcus aureus DNA polymerase III: design, synthesis, and biological evaluation. 1272 46

8-oxo-7,8-dihydroguanine (8-oxo-G) in DNA is a mutagenic adduct formed by reactive oxygen species. In Escherichia coli, 2,6-dihydroxy-5N-formamidopyrimidine (Fapy)-DNA glycosylase (Fpg) removes this mutagenic adduct from DNA. In this report, we demonstrate base excision repair (BER) synthesis of DNA containing 8-oxo-G with Fpg in vitro. Fpg cut the oligonucleotide at the site of 8-oxo-G, producing one nucleotide gap with 3' and 5' phosphate termini. Next, 3' phosphatase(s) in the supernatant obtained by precipitating a crude extract of E. coli with 40% ammonium sulfate, removed the 3' phosphate group at the gap, thus exposing the 3' hydroxyl group to prime DNA synthesis. DNA polymerase and DNA ligase then completed the repair. These results indicate the biological significance of the glycosylase and apurinic/ apyrimidinic (AP) lyase activities of Fpg, in concert with 3' phosphatase(s) to create an appropriately gapped substrate for efficient BER synthesis of DNA containing 8-oxo-G.
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PMID:Base excision repair synthesis of DNA containing 8-oxoguanine in Escherichia coli. 1275 14

Previous studies have shown that poly (ADP-ribose) polymerase (PARP) and DNA polymerase beta, nuclear enzymes, are associated with cell replication and DNA repair. The present study tests the hypothesis that hypoxia results in increased PARP and DNA polymerase activity in cerebral cortical neuronal nuclei to repair the hypoxia-induced damage to genomic DNA. Studies were conducted in 13 anesthetized and ventilated newborn piglets (age 3-5 days) divided into normoxic (n=5) and hypoxic (n=8) groups. Hypoxia was induced by decreasing inspired oxygen from 21% to 7% for 60 min. Cerebral tissue hypoxia was documented biochemically by determining the tissue levels of ATP and phosphocreatine (PCr). Following isolation of the cortical neuronal nuclei, the activity of PARP and DNA polymerase beta was determined. During hypoxia, the tissue ATP level decreased by 73% from 4.12+/-0.67 micromol/g brain to 1.12+/-0.34 micromol/g brain, and PCr decreased by 78% from 4.14+/-0.68-0.90+/-0.20 micromol/g brain. In hypoxic neuronal nuclei, PARP activity significantly increased from 5.88+/-0.51 pmol NAD/mg protein/h in normoxic nuclei to 10.04+/-2.02 (P=0.001). PARP activity inversely correlated with tissue ATP (r=0.78) and PCr levels (r=0.81). Administration of N-nitro-L-arginine prior to hypoxia decreased the hypoxia-induced increase in PARP activity by 67%. Endogenous DNA polymerase beta activity increased from 0.96+/-0.13 in normoxic nuclei to 1.39+/-0.18 nmol/mg protein/h in hypoxic nuclei (P<0.005). DNA polymerase beta activity in the presence of exogenous template increased from 1.54+/-0.14 in the normoxic to 2.42+/-0.26 nmol/mg protein/h in the hypoxic group (P<0.005). DNA polymerase beta activity in the presence or absence of template inversely correlated with the tissue ATP (r=0.95 and 0.84, respectively) and PCr levels (r=0.93 and 0.77, respectively). These results demonstrate that the activity of PARP and DNA polymerase beta enzymes increase with the increase in degree of cerebral tissue hypoxia. Furthermore, the results demonstrate a direct correlation between the PARP and the DNA polymerase beta activity. We conclude that tissue hypoxia results in increased PARP and DNA polymerase beta activities indicating activation of DNA repair mechanisms that may result in potential neuronal recovery following hypoxia and the hypoxia-induced increase in PARP activity is NO-mediated.
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PMID:Hypoxia-induced modification of poly (ADP-ribose) polymerase and dna polymerase beta activity in cerebral cortical nuclei of newborn piglets: role of nitric oxide. 1283 61

The thymidine 5'-triphosphate analogue containing a methylene group in place of the 5' oxygen atom can be prepared using modifications of published procedures and can substitute for the natural thymidine triphosphate in chain extension reactions catalyzed by Moloney-MLV reverse transcriptase. Using rabbit beta-globin mRNA as the template together with an appropriate primer, the enzyme readily makes full-length DNA transcripts in which all thymidine 5' oxygen atoms have been replaced with methylene groups. In sequence analyses using the partial depurination procedure, the analogue DNA transcript produces electrophoretic gel patterns identical with those of the corresponding natural DNA transcript. Experiments on second strand synthesis using the four regular triphosphates show that the analogue DNA transcript, like the natural transcript, can serve as a template. The two DNA duplexes (natural/natural and analogue/natural) formed by these reactions produce similar electrophoretic cleavage patterns when treated with either of the endonucleases HaeIII and EcoRI. However, further studies on template properties indicate that, while the enzyme makes a full-length product when using the analogue substrate with a natural DNA strand as template, it appears unable to use the analogue transcript as template with the analogue triphosphate as substrate during second strand synthesis. Preliminary experiments have also been carried out with a DNA polymerase. No products are detected reactions using Taq polymerase with PCR protocols containing the analogue triphosphate as the only source of thymidine.
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PMID:The analogue of thymidine triphosphate containing a methylene group in place of the 5' oxygen can serve as a substrate for reverse transcriptase. 1288 22

Oxidative damage to DNA by endogenous and exogenous reactive oxygen species has been directly linked to cancer, aging, and a variety of neurological disorders. The potential mutagenicity of the primary guanine oxidation product 8-oxo-7,8-dihydroguanine (Og) has been studied intensively, and much information is available about its miscoding potential in vitro and in vivo. Recently, a variety of DNA lesions have been identified as oxidation products of both guanine and 8-oxoguanine, among them spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). To address questions concerning the mutagenic potential of these secondary products of guanine oxidation, the effect of the lesions on proofreading by DNA polymerase was studied in vitro using the Klenow fragment of Escherichia coli polymerase I (Kf exo+). For the first time, k(cat)/K(m) values were obtained for proofreading of the X:N mismatches (X = Og, Gh, or Sp; N = A, G, or C). Proofreading studies of the terminal mismatches demonstrated the significance of the sequence context flanking the lesion on the 3' side. In addition, a sequence dependence was observed for Gh based on the identity of the base on the 5' side of the lesion providing evidence for a primer slippage mode if N was complementary to the 5' base. Internal mismatches were recognized by Kf exo+ resulting in the excision of the correct base pairs flanking mismatches from the 5' side. The absence of a sequence effect for the Gh- and Sp-containing duplexes can be attributed to the severe destabilization of the lesion-containing duplexes that promotes interaction with the exonuclease domain of the Klenow fragment.
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PMID:Effect of the oxidized guanosine lesions spiroiminodihydantoin and guanidinohydantoin on proofreading by Escherichia coli DNA polymerase I (Klenow fragment) in different sequence contexts. 1459 16

Radical oxygen species (ROS) generate various modified DNA bases. Among them 8-oxo-7,8-dihydroguanine (8oxoG) is the most abundant and seems to play a major role in mutagenesis and in carcinogenesis. 8oxoG is removed from DNA by the specific glycosylase OGG1. An additional post-replication repair is needed to correct the 8oxoG/A mismatches that are produced by persistent 8oxoG residues. This review is focused on the mechanisms of base excision repair (BER) of this oxidized base. It is shown that, in vitro, efficient and complete repair of 8oxoG/C pairs requires a core of four proteins, namely OGG1, APE1, DNA polymerase (Pol) beta, and DNA ligase I. Repair occurs predominantly by one nucleotide replacement reactions (short-patch BER) and Pol beta is the polymerase of election for the resynthesis step. However, alternative mechanisms can act on 8oxoG residues since Pol beta-null cells are able to repair these lesions. 8oxoG/A mismatches are repaired by human cell extracts via two BER events which occur sequentially on the two strands. The removal of the mismatched adenine is followed by preferential insertion of a cytosine leading to the formation of 8oxoG/C pairs which are then corrected by OGG1-mediated BER. Both repair events are inhibited by aphidicolin, suggesting that a replicative DNA polymerase is involved in the repair synthesis step. We propose that Pol delta/epsilon-mediated BER (long-patch BER) is the mode of repair when lesions persist or are formed at replication. Finally, we address the issues of the relative contribution of the two BER pathways to oxidative damage repair in vivo and the possible role of BER gene variants as cancer susceptibility genes.
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PMID:8-Oxoguanine DNA damage: at the crossroad of alternative repair pathways. 1463 50


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