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 family Poxviridae contains two subfamilies: the Entomopoxvirinae (poxviruses of insects) and the Chordopoxvirinae (poxviruses of vertebrates). Here we present the first characterization of the genome of an entomopoxvirus (EPV) which infects the North American migratory grasshopper Melanoplus sanguinipes and other important orthopteran pests. The 236-kbp M. sanguinipes EPV (MsEPV) genome consists of a central coding region bounded by 7-kbp inverted terminal repeats and contains 267 open reading frames (ORFs), of which 107 exhibit similarity to previously described genes. The presence of genes not previously described in poxviruses, and in some cases in any other known virus, suggests significant viral adaptation to the arthropod host and the external environment. Genes predicting interactions with host cellular mechanisms include homologues of the inhibitor of apoptosis protein, stress response protein phosphatase 2C, extracellular matrixin metalloproteases, ubiquitin, calcium binding EF-hand protein, glycosyltransferase, and a triacylglyceride lipase. MsEPV genes with putative functions in prevention and repair of DNA damage include a complete base excision repair pathway (uracil DNA glycosylase, AP endonuclease, DNA polymerase beta, and an NAD+-dependent DNA ligase), a photoreactivation repair pathway (cyclobutane pyrimidine dimer photolyase), a LINE-type reverse transcriptase, and a mutT homologue. The presence of these specific repair pathways may represent viral adaptation for repair of environmentally induced DNA damage. The absence of previously described poxvirus enzymes involved in nucleotide metabolism and the presence of a novel thymidylate synthase homologue suggest that MsEPV is heavily reliant on host cell nucleotide pools and the de novo nucleotide biosynthesis pathway. MsEPV and lepidopteran genus B EPVs lack genome colinearity and exhibit a low level of amino acid identity among homologous genes (20 to 59%), perhaps reflecting a significant evolutionary distance between lepidopteran and orthopteran viruses. Divergence between MsEPV and the Chordopoxvirinae is indicated by the presence of only 49 identifiable chordopoxvirus homologues, low-level amino acid identity among these genes (20 to 48%), and the presence in MsEPV of 43 novel ORFs in five gene families. Genes common to both poxvirus subfamilies, which include those encoding enzymes involved in RNA transcription and modification, DNA replication, protein processing, virion assembly, and virion structural proteins, define the genetic core of the Poxviridae.
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PMID:The genome of Melanoplus sanguinipes entomopoxvirus. 984 59

Two enzymes of base excision repair (BER), uracil DNA glycosylase (UDG) and DNA polymerase beta (beta pol), from HeLa cells co-eluted from Superose 12 FPLC columns. The UDG was completely displaced from 150-180-kDa fractions to 30- 70-kDa fractions by brief treatment with 0.5 N NaCl, pH 3.0, as expected when protein-protein associations are disrupted, but beta pol was not displaced by this treatment. UDG was not essential to the presence of beta pol in the 150-180-kDa enzyme complex. beta pol and UDG apparently reside in separate but co-eluting structures. Immunoaffinity chromatography showed that the association of UDG and beta pol was accounted for by attachment in common to DNA and that the association was abolished by eliminating DNA. Evidence for base excision repairosomes containing UDG and beta pol in protein-protein assemblies was not found. However, UDG and human AP endonuclease (HAP1) were associated with HSP70 and HSP27, which are present in 150-180-kDa and 30-70-kDa proteins of cell sonicates. The association of HSPs with BER enzymes was confirmed by hydroxyl radical protein-protein footprinting and immunoaffinity tests. The association of HSPs and BER enzymes is a novel finding. HSP binding may account for the presence of BER enzymes in the two large size class fractions and HSPs may have functional roles in BER.
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PMID:Heat-shock proteins associated with base excision repair enzymes in HeLa cells. 1062 18

Mammalian cells repair apurinic/apyrimidinic (AP) sites in DNA by two distinct pathways: a polymerase beta (pol beta)-dependent, short- (one nucleotide) patch base excision repair (BER) pathway, which is the major route, and a PCNA-dependent, long- (several nucleotide) patch BER pathway. The ability of a cell-free lysate prepared from asexual Plasmodium falciparum malaria parasites to remove uracil and repair AP sites in a variety of DNA substrates was investigated. We found that the lysate contained uracil DNA glycosylase, AP endonuclease, DNA polymerase, flap endonuclease, and DNA ligase activities. This cell-free lysate effectively repaired a regular or synthetic AP site on a covalently closed circular (ccc) duplex plasmid molecule or a long (382 bp), linear duplex DNA fragment, or a regular or reduced AP site in short (28 bp), duplex oligonucleotides. Repair of the AP sites in the various DNA substrates involved a long-patch BER pathway. This biology is different from mammalian cells, yeast, Xenopus, and Escherichia coli, which predominantly repair AP sites by a one-nucleotide patch BER pathway. The apparent absence of a short-patch BER pathway in P. falciparum may provide opportunities to develop antimalarial chemotherapeutic strategies for selectively damaging the parasites in vivo and will allow the characterization of the long-patch BER pathway without having to knock-out or inactivate a short-patch BER pathway, which is necessary in mammalian cells.
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PMID:DNA base excision repair in human malaria parasites is predominantly by a long-patch pathway. 1065 42

p12(DOC-1) is a growth suppressor identified and isolated from normal keratinocytes. Ectopic expression of p12(DOC-1) in squamous carcinoma cells led to the reversion of in vitro transformation phenotypes including anchorage independence, doubling time, and morphology. Here we report that p12(DOC-1) associates with DNA polymerase alpha/primase (pol-alpha:primase) in vitro and in cells. The pol-alpha:primase binding domain in p12(DOC-1) is mapped to the amino-terminal six amino acid (MSYKPN). The biological effect of p12(DOC-1) on pol-alpha:primase was examined using in vitro DNA replication assays. Using the SV40 DNA replication assay, p12(DOC-1) suppresses DNA replication, leveling at approximately 50%. Similar results were obtained using the M13 single-stranded DNA synthesis assay. Analysis of the DNA replication products revealed that p12(DOC-1) affects the initiation step, not the elongation phase. The p12(DOC-1) suppression of DNA replication is likely to be mediated either by a direct inhibitory effect on pol-alpha:primase or by its effect on cyclin-dependent kinase 2 (CDK2), a recently identified p12(DOC-1)-associated protein known to stimulate DNA replication by phosphorylating pol-alpha:primase. p12(DOC-1) suppresses CDK2-mediated phosphorylation of pol-alpha:primase. These data support a role of p12(DOC-1) as a regulator of DNA replication by direct inhibition of pol-alpha:primase or by negatively regulating the CDK2-mediated phosphorylation of pol-alpha:primase.
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PMID:p12(DOC-1), a growth suppressor, associates with DNA polymerase alpha/primase. 1087 24

Uracil, a promutagenic base in DNA can arise by spontaneous deamination of cytosine or incorporation of dUMP by DNA polymerase. Uracil is removed from DNA by uracil DNA glycosylase (UDG), the first enzyme in the uracil excision repair pathway. We recently reported that the Escherichia coli single-stranded DNA binding protein (SSB) facilitated uracil excision from certain structured substrates by E. coli UDG (EcoUDG) and suggested the existence of interaction between SSB and UDG. In this study, we have made use of the chimeric proteins obtained by fusion of N- and C-terminal domains of SSBs from E. coli and Mycobacterium tuberculosis to investigate interactions between SSBs and UDGs. The EcoSSB or a chimera containing its C-terminal domain interacts with EcoUDG in a binary (SSB-UDG) or a ternary (DNA-SSB-UDG) complex. However, the chimera containing the N-terminal domain from EcoSSB showed no interactions with EcoUDG. Thus, the C-terminal domain (48 amino acids) of EcoSSB is necessary and sufficient for interaction with EcoUDG. The data also suggest that the C-terminal domain (34 amino acids) of MtuSSB is a predominant determinant for mediating its interaction with MtuUDG. The mechanism of how the interactions between SSB and UDG could be important in uracil excision repair pathway has been discussed.
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PMID:Chimeras between single-stranded DNA-binding proteins from Escherichia coli and Mycobacterium tuberculosis reveal that their C-terminal domains interact with uracil DNA glycosylases. 1127 60

Nucleoside analogues such as acyclovir and ganciclovir have been the mainstay of therapy for alphaherpesviruses (herpes simplex virus (HSV) and varicella-zoster virus (VZV)) and cytomegalovirus (CMV) infections, respectively. Drug-resistant herpesviruses are found relatively frequently in the clinic, almost exclusively among severely immunocompromised patients receiving prolonged antiviral therapy. For instance, close to 10% of patients with AIDS receiving intravenous ganciclovir for 3 months excrete a drug-resistant CMV isolate in their blood or urine and this percentage increases with cumulative drug exposure. Many studies have reported that at least some of the drug-resistant herpesviruses retain their pathogenicity and can be associated with progressive or relapsing disease. Viral mutations conferring resistance to nucleoside analogues have been found in either the drug activating/phosphorylating genes (HSV or VZV thymidine kinase, CMV UL97 kinase) and/or in conserved regions of the viral DNA polymerase. Currently available second line agents for the treatment of herpesvirus infections--the pyrophosphate analogue foscarnet and the acyclic nucleoside phosphonate derivative cidofovir--also inhibit the viral DNA polymerase but are not dependent on prior viral-specific activation. Hence, viral DNA polymerase mutations may lead to a variety of drug resistance patterns which are not totally predictable at the moment due to insufficient information on specific drug binding sites on the polymerase. Although some CMV and HSV DNA polymerase mutants have been found to replicate less efficiently in cell cultures, further research is needed to correlate viral fitness and clinical outcome.
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PMID:Resistance of herpesviruses to antiviral drugs: clinical impacts and molecular mechanisms. 1213 84

Azodicarbonamide tested as an anti-HIV agent was reported to expulse zinc from viral zinc-cysteine factors and to inhibit calcium mobilization machinery. It has structural analogy with hydroxyurea that inhibits ribonucleotide reductase and could also act on this target. Azodicarbonamide was therefore tested for its capacity to modulate deoxyribonucleotides triphosphate pools alone or in combination with other agents in the lymphoblastic SUP-T1 cell line susceptible to HIV infection. The deoxyribonucleotides triphosphate were evaluated by an enzymatic assay using sequenase. Two hours exposure of SUP-T1 cells to 100 microM azodicarbonamide induced a 50% reduction of each deoxyribonucleotide triphosphate. Among other inhibitors of nucleotide metabolism (hydroxyurea, methotrexate and thymidine), hydroxyurea only reproduces the effect of azodicarbonamide. This suggests, but does not demonstrate directly, that azodicarbonamide inhibits ribonucleotide reductase activity. The combination of azodicarbonamide with each of these inhibitors affected particularly the dCTP pool. During this study it was also suggested that azodicarbonamide could interfere with thymidine phosphorylation. Thymidine phosphorylating activity was measured with 3H-thymidine as substrate. In acellular preparations, azodicarbonamide also non-competitively inhibits thymidine phosphorylating activity. This effect was not reproduced by hydroxyurea. Thus, in vitro azodicarbonamide decreases the intracellular pool of deoxyribonucleotide and thymidine phosphorylation.
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PMID:Ribonucleotide reductase and thymidine phosphorylation: two potential targets of azodicarbonamide. 1214 96

The mechanism by which brief episodes of cerebral ischemia confer protection (tolerance) against subsequent prolonged ischemic challenges remains unclear, but may involve upregulation of cell injury repair capability. The mitochondrion is a key site for the regulation of cell death pathways, and damage to mitochondrial genes has been linked to a number of neurologic diseases and aging. Therefore, the authors examined the response of the DNA base excision repair (BER) pathway in rat brain mitochondria after either brief (tolerance-inducing) or prolonged (injury-producing) focal cerebral ischemia. Brief (30-minute) middle cerebral artery occlusion (MCAO) induced mild oxidative mitochondrial DNA damage and initiated a prolonged (up to 72-hour) activation above control levels of the principal enzymes of the mitochondrial BER pathway, including uracil DNA glycosylase, apurinic/apyrimidinic (AP) endonuclease, DNA polymerase-gamma, and DNA ligase. In contrast, prolonged (100-minute MCAO) ischemia induced more substantial mitochondrial oxidative DNA damage whereas elevation of BER activity was transient (approximately 1 hour), declining to less than control levels over the course of 4 to 72 hours. These data reveal the differences in BER capacity after brief or prolonged ischemia, which may contribute to the neuron's ability to resist subsequent ischemic insults.
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PMID:Upregulation of mitochondrial base-excision repair capability within rat brain after brief ischemia. 1250 94

Damaged DNA bases are removed from mammalian genomes by base excision repair (BER). Single nucleotide BER requires several enzymatic activities, including DNA polymerase and 5',2'-deoxyribose-5-phosphate lyase. Both activities are intrinsic to four human DNA polymerases whose base substitution error rate during gap-filling DNA synthesis varies by more than 10,000-fold. This suggests that BER fidelity could vary over a wide range in an enzyme dependent manner. To investigate this possibility, here we describe an assay to measure the fidelity of BER reactions reconstituted with purified enzymes. When human uracil DNA glycosylase, AP endonuclease, DNA polymerase beta, and DNA ligase 1 replace uracil opposite template A or G, base substitution error rates are <or=0.3 to <or=2.8 x 10-4. BER error rates are higher when excess incorrect dNTPs are included in the reaction or when wild type DNA polymerase beta is replaced by DNA polymerase beta variants that fill single nucleotide gaps with lower fidelity. Under these conditions, the base substitution fidelity of polymerase beta-dependent BER is 3-8-fold higher than is single nucleotide gap filling by polymerase beta alone. Thus other proteins in the BER reaction may enhance the base substitution fidelity of DNA polymerase beta during single nucleotide BER.
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PMID:The base substitution fidelity of DNA polymerase beta-dependent single nucleotide base excision repair. 1273 1

The majority of DNA in eukaryotic cells exists in the highly condensed structural hierarchy of chromatin, which presents a challenge to DNA repair enzymes in that recognition, incision, and restoration of the original sequence at most sites must take place within these structural constraints. To test base excision repair (BER) activities on chromatin substrates, an in vitro system was developed that uses human uracil DNA glycosylase (UDG), apyrimidinic/apurinic endonuclease (APE), and DNA polymerase beta (pol beta) on homogeneously damaged, rotationally positioned DNA in nucleosomes. We find that UDG and APE carry out their combined catalytic activities with reduced efficiency on nucleosome substrates ( approximately 10% of that on naked DNA). Furthermore, these enzymes distinguish between two different rotational settings of the lesion on the histone surface, showing a 2- to 3-fold difference in activity between uracil facing "toward" and "away from" the histones. However, UDG and APE will digest such substrates to completion in a concentration-dependent manner. Conversely, the synthesis activity of pol beta is inhibited completely by nucleosome substrates and is independent of enzyme concentration. These results suggest that the first two steps of BER, UDG and APE, may occur "unassisted" in chromatin, whereas downstream factors in this pathway (i.e., pol beta) may require nucleosome remodeling for efficient DNA BER in at least some regions of chromatin in eukaryotic cells.
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PMID:Suppressed catalytic activity of base excision repair enzymes on rotationally positioned uracil in nucleosomes. 1279 67


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