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)

We found that DNA polymerase I from Chlamydiophila pneumoniae AR39 (CpDNApolI) presents DNA-dependent DNA polymerase activity, but has no detectable 3' exonuclease activity. CpDNApolI-dependent DNA synthesis was performed using DNA templates carrying different lesions. DNAs containing 2'-deoxyuridine (dU), 2'-deoxyinosine (dI) or 2'-deoxy-8-oxo-guanosine (8-oxo-dG) served as templates as effectively as unmodified DNAs for CpDNApolI. Furthermore, the CpDNApolI could bypass natural apurinic/apyrimidinic sites (AP sites), deoxyribose (dR), and synthetic AP site tetrahydrofuran (THF). CpDNApolI could incorporate any dNMPs opposite both of dR and THF with the preference to dAMP-residue. CpDNApolI preferentially extended primer with 3'-dAMP opposite dR during DNA synthesis, however all four primers with various 3'-end nucleosides (dA, dT, dC, and dG) opposite THF could be extended by CpDNApolI. Efficiently bypassing of AP sites by CpDNApolI was hypothetically attributed to lack of 3' exonuclease activity.
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PMID:Chlamydial DNA polymerase I can bypass lesions in vitro. 1671 85

Crystallographic studies have implicated several residues of the p66 fingers subdomain of human immunodeficiency virus type-1 reverse transcriptase in contacting the single-stranded template overhang immediately ahead of the DNA polymerase catalytic center. This interaction presumably assists in inducing the appropriate geometry on the template base for efficient and accurate incorporation of the incoming dNTP. To investigate this, we introduced nucleoside analogs either individually or in tandem into the DNA template ahead of the catalytic center and investigated whether they induce pausing of the replication machinery before serving as the template base. Analogs included abasic tetrahydrofuran linkages, neutralizing methylphosphonate linkages, and conformationally locked nucleosides. In addition, several Phe-61 mutants were included in our analysis, based on previous data indicating that altering this residue affects both strand displacement synthesis and the fidelity of DNA synthesis. We demonstrate here that altering the topology of the template strand two nucleotides ahead of the catalytic center can interrupt DNA synthesis. Mutating Phe-61 to either Ala or Leu accentuates this defect, whereas replacement with an aromatic residue (Trp) allows the mutant enzyme to bypass the template analogs with relative ease.
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PMID:Examining interactions of HIV-1 reverse transcriptase with single-stranded template nucleotides by nucleoside analog interference. 1686 79

Mutations in the adenomatous polyposis coli (APC) gene are associated with an early onset of colorectal carcinogenesis. Previously, we described a novel role for the APC polypeptide in base excision repair (BER). The single-nucleotide (SN) and long-patch (LP) BER pathways act to repair the abasic sites in DNA that are induced by stressors, such as spontaneous oxidation/reduction, alkylation, and hyperthermia. We have shown that APC interacts with DNA polymerase beta (Pol-beta) and flap endonuclease 1 (Fen-1) and blocks Pol-beta-directed strand-displacement synthesis. In this study, we have mapped the APC interaction site in Pol-beta and have found that Thr79, Lys81, and Arg83 of Pol-beta were critical for its interaction with APC. The Pol-beta protein (T79A/K81A/R83A) blocked strand-displacement DNA synthesis in which tetrahydrofuran was used as DNA substrate. We further showed that the APC-mediated blockage of LP-BER was due to inhibition of Fen-1 activity. Analysis of the APC-mediated blockage of SN-BER indicated that the interaction of APC with Pol-beta blocked SN-BER activity by inhibiting Pol-beta-directed deoxyribose phosphate lyase activity. Collectively, our findings indicate that APC blocked both Pol-beta-directed SN- and LP-BER pathways and increased sensitivity of cells to alkylation induced DNA damage.
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PMID:Structure/function analysis of the interaction of adenomatous polyposis coli with DNA polymerase beta and its implications for base excision repair. 1799 39

DNA tandem lesions are comprised of two contiguously damaged nucleotides. This subset of clustered lesions is produced by a variety of oxidizing agents, including ionizing radiation. Clustered lesions can inhibit base excision repair (BER). We report the effects of tandem lesions composed of a thymine glycol and a 5'-adjacent 2-deoxyribonolactone (LTg) or tetrahydrofuran abasic site (FTg). Some BER enzymes that act on the respective isolated lesions do not accept the tandem lesion as a substrate. For instance, endonuclease III (Nth) does not excise thymine glycol (Tg) when it is part of either tandem lesion. Similarly, endonuclease IV (Nfo) does not incise L or F when they are in tandem with Tg. Long-patch BER overcomes inhibition by the tandem lesion. DNA polymerase beta (Pol beta) carries out strand displacement synthesis, following APE1 incision of the abasic site. Pol beta activity is enhanced by flap endonuclease (FEN1), which cleaves the resulting flap. The tandem lesion is also incised by the bacterial nucleotide excision repair system UvrABC with almost the same efficiency as an isolated Tg. These data reveal two solutions that DNA repair systems can use to counteract the formation of tandem lesions.
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PMID:DNA tandem lesion repair by strand displacement synthesis and nucleotide excision repair. 1834 Dec 93

Oxidative DNA damage incidental to normal respiratory metabolism poses a particular threat to genomes of highly metabolic-long lived cells. We show that post-mitotic brain has capacity to repair oxidatively damaged DNA ends, which are targets of the long patch (LP) base excision repair (BER) subpathway. LP-BER relies, in part, on proteins associated with DNA replication, including proliferating cell nuclear antigen and is inherent to proliferating cells. Nonetheless, repair products are generated with brain extracts, albeit at slow rates, in the case of 5'-DNA ends modeled with tetrahydrofuran (THF). THF at this position is refractory to DNA polymerase beta 5'-deoxyribose 5-phosphate lyase activity and drives repair into the LP-BER subpathway. Comparison of repair of 5'-THF-blocked termini in the post-mitotic rat brain and proliferative intestinal mucosa, revealed that in mucosa, resolution of damaged 5'-termini is accompanied by formation of larger repair products. In contrast, adducts targeted by the single nucleotide BER are proficiently repaired with both extracts. Our findings reveal mechanistic differences in BER processes selective for the brain versus proliferative tissues. The differences highlight the physiological relevance of the recently proposed 'Hit and Run' mechanism of alternating cleavage/synthesis steps, in the proliferating cell nuclear antigen-independent LP-BER process.
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PMID:DNA polymerase beta-catalyzed-PCNA independent long patch base excision repair synthesis: a mechanism for repair of oxidatively damaged DNA ends in post-mitotic brain. 1875 43

Under isothermal conditions, short oligodeoxynucleotides (ODNs) were elongated to long DNA by Vent(exo(-)), a thermophilic DNA polymerase, in the presence of dNTPs. Short ODNs (14-28 nt) were designed to form hairpin structures based on the sequence we obtained from de novo DNA synthesis in the presence of restriction enzyme Tsp509I. As short as 14-nt-long DNA could be elongated to longer than 20000 nucleotides by Vent(exo(-)) at 65 degrees C in 1 h. The high efficiency of elongation at very low concentration (<1 nM) supported the THF-SPE (terminal hairpin formation and self-priming extension) mechanism we purposed for DNA elongation during de novo DNA synthesis. The hairpin structure forms at a DNA duplex end as a self-priming complex, followed by strand displacement extension to longer DNA. The highly efficient elongation attributes to the successive repetition of the process of THF-SPE.
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PMID:Mechanism of DNA elongation during de novo DNA synthesis. 1877 28

A new approach that improves the efficiency and specificity of polymerase chain reaction (PCR) has been developed. Heat-sensitive 3'-protected derivatives of 2'-deoxyribonucleoside 5'-triphosphates (dNTPs) have been synthesized and used as substitutes for natural dTTP, dCTP, dATP, and dGTP in PCR. Since 3'-protected dNTPs are either nonsubstrates or terminating substrates for Taq DNA polymerase, they do not support primer extension/elongation at low stringency conditions during PCR sample preparation when PCR artifacts such as primer dimers and mispriming products can form. At the initial heat-denaturing step and during the PCR sequence, the 3'-protecting group is cleaved, releasing 3'-unprotected dNTP that is a natural substrate for DNA polymerase. As a result, the primer extension/elongation proceeds only at an elevated temperature of PCR, when the interaction of primers and template is highly stringent and specific. Several 3'-protecting groups covering a wide range of deprotection kinetics have been tested. The 3'-O-tetrahydrofuranyl derivatives of dNTPs have demonstrated the best properties leading to a drastically reduced accumulation of PCR artifacts, such as "primer dimers" and "mispriming" products. Overall, PCR with 3'-THF-protected dNTPs demonstrated substantially improved performance and was more efficient and specific compared to PCR with standard dNTPs.
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PMID:3'-Protected 2'-deoxynucleoside 5'-triphosphates as a tool for heat-triggered activation of polymerase chain reaction. 1943 48

To examine base excision repair (BER) capacity in the context of living cells, we developed and applied a plasmid-based reporter assay. Non-replicating plasmids containing unique DNA base lesions were designed to express luciferase only after lesion repair had occurred, and luciferase expression in transfected cells was measured continuously during a repair period of 14 h. Two types of DNA lesions were examined: uracil opposite T reflecting repair primarily by the single-nucleotide BER sub-pathway, and the abasic site analogue tetrahydrofuran (THF) opposite C reflecting repair by long-patch BER. We found that the repair capacity for uracil-DNA in wild type mouse fibroblasts was very strong, whereas the repair capacity for THF-DNA, although strong, was slightly weaker. Repair capacity in DNA polymerase beta (Pol beta) null cells for uracil-DNA and THF-DNA was reduced by approximately 15% and 20%, respectively, compared to that in wild type cells. In both cases, the repair deficiency was fully complemented in Pol beta null cells expressing recombinant Pol beta. The effect of inhibition of poly(ADP-ribose) polymerase (PARP) activity on repair capacity was examined by treatment of cells with the inhibitor 4-amino-1,8-naphthalimide (4-AN). PARP inhibition decreased the repair capacity for both lesions in wild type cells, and this reduction was to the same level as that seen in Pol beta null cells. In contrast, 4-AN had no effect on repair in Pol beta null cells. The results highlight that Pol beta and PARP function in the same repair pathway, but also suggest that there is repair independent of both Pol beta and PARP activities. Thus, before the BER capacity of a cell can be predicted or modulated, a better understanding of Pol beta and PARP activity-independent BER pathways is required.
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PMID:DNA polymerase beta and PARP activities in base excision repair in living cells. 1974 37

The efficiency of enzymatic conversion of DNA complexes containing non-nucleotide inserts has been studied. T4 DNA ligase and Taq DNA polymerase have been included in the study as examples of widely used DNA-dependent enzymes. A series of substrate DNA complexes have been formed using native oligonucleotides and bridged ones bearing non-nucleotide inserts based on phosphodiesters of di-, tetra-, or hexaethylene glycol, 1,5-pentanediol, 1,10-decanediol, and 3-hydroxy-2(hydroxymethyl)-tetrahydrofuran. The perturbation in DNA located far from the site of the enzyme action had almost no influence on the substrate properties of the complex, while insertion near this site significantly deteriorated them. The use of a series of modified duplexes allows one to locate the position of the enzyme-binding site on DNA substrate with the accuracy of 1-2 nucleotides. The presence of a non-nucleotide insert in the complex has been also shown to enhance the efficiency of single mismatch discrimination upon both template-directed ligation and extension of oligonucleotides.
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PMID:Bridged oligonucleotides as molecular probes for investigation of enzyme-substrate interaction and allele-specific analysis of DNA. 1991 12

N3-methyladenine (3-mA) is a cytotoxic lesion formed by the reaction of DNA with many methylating agents, including antineoplastic drugs, environmental agents and endogenously generated compounds. The toxicity of 3-mA has been attributed to its ability to block DNA polymerization. Using Me-lex, a compound that selectively and efficiently reacts with DNA to afford 3-mA, we have observed in yeast a mutational hotspot at the 5'-terminus of an A(4) tract. In order to explore the potential role of sequence-dependent DNA polymerase bypass of 3-mA, we developed an in vitro system to prepare 3-mA modified substrates using Me-lex. We detail the effects of 3-mA, its stable isostere analogue, 3-methyl-3-deazaadenine, 3-deazaadenine and an THF abasic site on DNA polymerization within an A(4) sequence. The methyl group on 3-mA and 3-methyl-3-deazaadenine has a pronounced inhibitory effect on DNA polymerization. There was no sequence selectivity for the bypass of any of the lesions, except for the abasic site, which was most efficiently by-passed when it was on the 5'-terminus of the A(4) tract. The results indicate that the weak mutational pattern induced by Me-lex may result form the depurination of 3-mA to an abasic site that is bypassed in a sequence dependent context.
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PMID:Effect of n3-methyladenine and an isosteric stable analogue on DNA polymerization. 2093 69


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