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)

Four isomeric benzo[a]pyrene-deoxyadenosine adducts, corresponding to the products of trans opening of the epoxide ring in the four configurationally isomeric benzo[a]pyrene dihydrodiol epoxides by the amino group of deoxyadenosine, were separately introduced into each of two 16-mer sequence contexts. The sequences were from the supF gene, and the site of the adducted adenine was known, for some hydrocarbon dihydrodiol epoxides, to be a hotspot for mutation in Context I and a coldspot for mutation in Context II. Using primers complementary to the 3' ends of these oligonucleotides, the abilities of several polymerases to replicate these templates in vitro were investigated. Each adduct proved to be an effective block to primer extension such that only with high concentrations of exo- Klenow fragment was any bypass of adducts seen. DNA polymerase alpha and HIV-1 reverse transcriptase were blocked 3' to the adduct when the configuration at C10 of the hdyrocarbon was S, and some introduction of thymine opposite the adenine adduct was seen with the R configuration. Incorporation of a nucleotide opposite the adduct occurred more readily with Sequenase and the Klenow fragment, and the mutagenic introduction of adenine was apparent in most cases. This corresponded to the A-->T transversions frequently seen in mutation studies with hydrocarbon dihydrodiol epoxides that react extensively with adenine in DNA. Overall, it was clear that sequence context, adduct stereochemistry, and the choice of polymerase all influenced the polymerization reaction. With these in vitro systems, no major differences correlating with the differing tumorigenicities of the isomeric dihydrodiol epoxides or with the hotspot or coldspot nature of the sequences were detected.
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PMID:Primer extension by various polymerases using oligonucleotide templates containing stereoisomeric benzo[a]pyrene-deoxyadenosine adducts. 752 75

Calcium and its receptor protein calmodulin function in the regulation of proliferation of mammalian cells. A 68 kDa calmodulin-specific binding protein was shown previously to be associated with growth factor-dependent progression of a variety of mammalian cells from G1 to S phase and to stimulate DNA synthesis in permeabilized hematopoietic progenitor cells. In this report we show that the 68 kDa calmodulin-specific binding protein in HeLa cells is tightly associated with the DNA polymerase alpha-primase component of the 21S complex of enzymes for DNA synthesis. The 68 kDa calmodulin-binding protein and the DNA polymerase alpha-primase complex cofractionate during Q-Sepharose chromatography to isolate the 21S enzyme complex, native and denatured DNA-cellulose to dissociate the 21S complex, and DEAE-Bio-Gel chromatography to isolate the multiprotein DNA polymerase alpha-primase complex. The 68 kDa calmodulin-specific binding protein and DNA polymerase alpha also bind and coelute during affinity chromatography on calmodulin-agarose. They also coprecipitate with C10-agarose-linked monoclonal antibody SJK 132-20 to human DNA polymerase alpha. The tight association of the 68 kDa calmodulin-binding protein to the DNA polymerase alpha-primase complex supports a function for this protein in the regulation of DNA synthesis in vivo.
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PMID:The 68 kDa calmodulin-binding protein is tightly associated with the multiprotein DNA polymerase alpha-primase complex in HeLa cells. 769 50

DNA adducts of the environmental carcinogen benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) interact stereospecifically with prokaryotic and eukaryotic polymerases in vitro. Toward understanding the capacity to replicate past different diastereomers of BPDE at specific sites in DNA, six deoxyoligonucleotides, each 33 bases long, were constructed with stereochemically defined BPDE adducts on adenine N6 at position two of the human N-ras codon 61. Four polymerases that were studied under single encounters with the template-primer complex terminated synthesis one base 3' to the lesion with all the adducted templates. When multiple encounters between polymerase and substrate were permitted, each of the polymerases analyzed revealed a unique pattern for a given adducted template. The general replication pattern was encompassed under two categories, reflecting the significance of the R and S configurations of C10 of the pyrenyl ring attached to the single-stranded DNA template. Furthermore, within each of these categories, every polymerase demonstrated distinct quantitative differences in product accumulation at a given site, for the various adducted templates. Among the polymerases utilized in this study, exonuclease-deficient Klenow fragment of polymerase I (exo- KF) exhibited the most efficient translesion synthesis resulting in approximately 16% full-length products with the modified templates bearing adducts with C10-S configuration. In contrast, chain elongation with bacteriophage T4 DNA polymerase bearing an active 3'-->5' exonucleolytic activity was most strongly inhibited by all six BPDE-adducted templates. Misincorporation of A opposite the adduct occurred in all the templates when polymerized with Sequenase, whereas exo- KF preferentially incorporated C opposite the C10-R BPDE adducts and A opposite the C10-S BPDE adducts.
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PMID:In vitro replication by prokaryotic and eukaryotic polymerases on DNA templates containing site-specific and stereospecific benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide adducts. 775 32

DNA structural perturbations that are induced by site specifically and stereospecifically defined benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) adducts are directly correlated with mutagenesis, leading to cellular transformation. Although previous investigations had established that replication of DNAs containing N(6) -BPDE dA adducts at the second position in the N-ras codon 61(CAA) (61(2) ) resulted exclusively in A to G transitions, NMR analyses not only established the structural basis for this transition mutation but also predicted that if the adduct were positioned at the third position in the same codon, an expanded spectra of mutations was possible. To test this prediction, replication of DNAs containing C10 S-BPDE and C10 R-BPDE lesions linked through the N(6) position of adenine in the sequence context N-ras codon 61, position 3 (C10 S-BPDE and C10 R-BPDE at 61(3) ) was carried out in Escherichia coli, and these data revealed a wide mutation spectrum. In addition to A to G transitions produced by replication of both lesions, replication of the C10 S-BPDE and C10 R-BPDE adducts also yielded A to C and A to T transversions, respectively. Analyses of single nucleotide incorporation using Sequenase 2.0 and exonuclease-deficient E. coli Klenow fragment and pol II not only revealed high fidelity synthesis but also demonstrated the same hierarchy of preference opposite a particular lesion, independent of the sequence context. Primer extension assays with the two lesions at N-ras 61(3) resulted in truncated products, with the C10 S-BPDE adducts being more blocking than C10 R-BPDE lesions, and termination of synthesis was more pronounced at position 61(3) than at 61(2) for each of the lesions.
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PMID:Sequence context modulation of polycyclic aromatic hydrocarbon-induced mutagenesis. 2391 16

The enhancement of heat stress tolerance in crops is an important challenge for food security to facilitate adaptation to global warming. In Arabidopsis thaliana, the transcriptional regulator DNA polymerase II subunit B3-1 (DPB3-1)/nuclear factor Y subunit C10 (NF-YC10) has been reported as a positive regulator of Dehydration-responsive element binding protein 2A (DREB2A), and the overexpression of DPB3-1 enhances heat stress tolerance without growth retardation. Here, we show that DPB3-1 interacts with DREB2A homologues in rice and soya bean. Transactivation analyses with Arabidopsis and rice mesophyll protoplasts indicate that DPB3-1 and its rice homologue OsDPB3-2 function as positive regulators of DREB2A homologues. Overexpression of DPB3-1 did not affect plant growth or yield in rice under nonstress conditions. Moreover, DPB3-1-overexpressing rice showed enhanced heat stress tolerance. Microarray analysis revealed that many heat stress-inducible genes were up-regulated in DPB3-1-overexpressing rice under heat stress conditions. However, the overexpression of DPB3-1 using a constitutive promoter had almost no effect on the expression of these genes under nonstress conditions. This may be because DPB3-1 is a coactivator and thus lacks inherent transcriptional activity. We conclude that DPB3-1, a coactivator that functions specifically under abiotic stress conditions, could be utilized to increase heat stress tolerance in crops without negative effects on vegetative and reproductive growth.
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PMID:The Arabidopsis transcriptional regulator DPB3-1 enhances heat stress tolerance without growth retardation in rice. 2684 Nov 13

Egg drop syndrome virus (EDSV) can markedly decrease egg production in laying hens. Duck is the natural host of EDSV. EDSV derived from ducks abrogate egg drop in laying hens. We have previously confirmed that duck-derived EDSVs have a variety of replication activities in chick embryo liver (CEL) cells. However, it is currently unclear whether duck-derived EDSV could display tropism and adaptation in laying hens. This study assessed whether duck-derived EDSV can adapt to laying hens, and estimated the inducing factors. Complete genome sequences of duck-derived EDSVs (D11-JW-012, D11-JW-017, and D11-JW-032 isolates) with various replication efficiency in CEL cells and C10-GY-001 isolate causing disease in laying hens were analyzed to find their differences. Phylogenetic analysis of complete genome sequence revealed that C10-GY-001, D11-JW-032, and strain 127 virus as vaccine were clustered into the same group, with D11-JW-012 and D11-JW-017 clustered in another group. Comparison between D11-JW-012 isolate that poorly replicated and D11-JW-017 isolate that replicated well in CEL cells in same cluster revealed six amino acid differences on IVa2, DNA polymerase, endopeptidase, and DNA-binding protein. These amino acids might be key candidates enhancing cellular tropism in chicken. When the pathogenicities of these isolates in laying hens were compared, D11-JW-032 showed severe signs similar to 127 virus, D11-JW-017 showed intermediate signs, while D11-JW-012 showed almost no sign. Eleven amino acids differed between D11-JW-032 and D11-JW-017, and 17 amino acids were different between D11-JW-032 and D11-JW-012. These results suggest that EDSVs derived from ducks have various pathogenicities in laying hens. Key amino acid candidates might have altered their affinity to tropism of laying hens, causing difference pathogenicities.
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PMID:Tropism and infectivity of duck-derived egg drop syndrome virus in chickens. 2848 13

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