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)

A bovine genomic DNA library was surveyed with respect to the template activity for RNA-primed DNA synthesis by calf thymus DNA polymerase alpha-primase complex. About 7% of the single-stranded DNA clones contained distinct initiation sites consisting of pyrimidine clusters of pyrimidine-rich sequences. The initiation sites were located at or near the 3'-end of the pyrimidine clusters. One of these sequences, containing a 10-mer pyrimidine cluster with major initiation sites, was analyzed in detail. By the successive substitutions of pyrimidines in the cluster with oligodeoxydenylate [(dA)n] in the 5' to 3' direction, the minimum length of the initiation sequence was estimated to be as long as the 7-mer. In contrast, when one or three pyrimidines at the 3'-end of the cluster were replaced with (dA)n, the priming activity was largely lost, indicating that these pyrimidine residues were indispensable for priming. Furthermore, base substitutions of upstream or downstream sequences outside the pyrimidine cluster also decreased the total priming frequencies. Interestingly, the base substitutions inside or outside of the pyrimidine cluster sometimes caused a shift in the major priming sites. These results indicate that the minimum priming unit of the CTPPS1 template for RNA-primed DNA synthesis consists of a pyrimidine cluster (6-mer) with one purine at its 3'-border and that both the 3'-downstream 6-bases and the 5'-upstream 17-bases modulate the priming by enhancing the priming frequency and/or slightly shifting the sites of initiation of primer synthesis. It was also revealed that the lengths of the product RNA primers became shorter as the length of pyrimidine cluster was shortened by substitution with (dA)n. The gel retardation assay further showed that the complex formation between DNA polymerase alpha-primase and the DNA templates was strongly in competition with poly(dC), poly(dG), and poly(dT) but not with poly(dA). Furthermore, template activities as well as the pyrimidine contents of a series of base-substituted DNA correlated well with their affinities to the enzyme, as measured by both gel retardation assay and their Km values for the priming reaction. Apparently, DNA polymerase alpha-primase primarily recognizes the minimum priming unit consisting of a pyrimidine cluster with a purine at the 3'-boundary of purine, but the initiation of primer RNA synthesis can be modified by pyrimidine residues outside of the minimum priming unit.
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PMID:RNA priming coupled with DNA synthesis on natural template by calf thymus DNA polymerase alpha-primase. 750 26

A recombinant human AP endonuclease, HAP1, was constructed and characterized with respect to its ability to recognize and act upon a model double-stranded 39-mer oligodeoxyribonucleotide substrate containing a strand break site with 3'-phosphoglycolate and 5'-phosphate end-group chemistries. This oligodeoxyribonucleotide substrate exactly duplicates the chemistry and configuration of a major DNA lesion produced by ionizing radiation. HAP1 was found to recognize the strand break, and catalyze the release of the 3'-phosphoglycolate as free phosphoglycolic acid. The enzyme had a Vmax of 0.1 fmole/min/pg of HAP1 protein, and a Km of 0.05 microM for the 3'-phosphoglycolate strand break lesion. The mechanism of catalysis was hydrolysis of the phosphate ester bond between the 3'-phosphoglycolate moiety and the 3'-carbon of the adjacent dGMP moiety within the oligonucleotide. The resulting DNA contained a 3'-hydroxyl which supported nucleotide incorporation by E. coli DNA polymerase I large fragment. AP endonucleolytic activity of HAP1 was examined using an analogous double-stranded 39-mer oligodeoxyribonucleotide substrate, in which the strand break site was replaced by an apyrimidinic site. The Vmax and Km for the AP endonuclease reaction were 68 fmole/min/pg of HAP1 protein and 0.23 microM, respectively.
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PMID:Removal of 3'-phosphoglycolate from DNA strand-break damage in an oligonucleotide substrate by recombinant human apurinic/apyrimidinic endonuclease 1. 751 64

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

The cytotoxicity of chloropolypyridyl ruthenium complexes of structural formulas [Ru(terpy)-(bpy)Cl]Cl, cis-[Ru(bpy)2Cl2], and mer-[Ru(terpy)Cl3] (terpy = 2,2':6'2"-terpyridine, bpy = 2,2'-bipyridyl) has been studied in murine and human tumor cell lines. The results show that mer-[Ru(terpy)Cl3] exhibits a remarkably higher cytotoxicity than the other complexes. Moreover, investigations of antitumor activity in a standard tumor screen have revealed the highest efficiency for mer-[Ru(terpy)Cl3]. In a cell-free medium, the ruthenium complexes coordinate to DNA preferentially at guanine residues. The resulting adducts can terminate DNA synthesis by thermostable VentR DNA polymerase. The reactivity of the complexes to DNA, their efficiency to unwind closed, negatively supercoiled DNA, and a sequence preference of their DNA adducts (studied by means of replication mapping) do not show a correlation with biological activity. On the other hand, the cytotoxic mer-[Ru(terpy)Cl3] exhibits a significant DNA interstrand cross-linking, in contrast to the inactive complexes which exhibit no such efficacy. The results point to a potential new class of metal-based antitumor compounds acting by a mechanism involving DNA interstrand cross-linking.
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PMID:Correlation between cytotoxicity and DNA binding of polypyridyl ruthenium complexes. 754 81

DNA polymorphisms generated by the random amplified polymorphic DNA polymerase chain reaction (RAPD PCR) were used to analyse 41 isolates investigated in the European Fusarium sambucinum Project (EFSP). Employing ten arbitrary (10-mer) oigonucleotides and simple repeat sequences (M13, (GACA)4) as single primers, informative banding patterns typical for identifying European populations of Fusarium sambucinum Fuckel s. str., F. torulosum (Berk. & Curt.) Nirenberg and F. venenatum Nirenberg were obtained.
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PMID:Differentiation of Fusarium sambucinum Fuckel sensu lato and related species by RAPD PCR. 756 52

In order to examine the effect of adenine N6 adducts of styrene oxide (SO) on DNA replication, 33-mer templates were constructed bearing site-specific and stereospecific SO modifications. Both R- and S-SO adducts were introduced at four different base positions within a sequence containing codons 60-62 from the human N-ras gene. The resulting eight templates were replicated in primer extension assays using the Klenow fragment, Sequenase 2.0, T4 polymerase holoenzyme, polymerase alpha, and polymerase beta. Replication of the damaged templates was analyzed under conditions defining single and/or multiple encounters between the polymerase and the substrate. Polymerization by all five enzymes was sensitive to both the local sequence context and the chirality of the SO adduct. For example, R-SO lesions placed at the third position of N-ras codon 61 were readily bypassed, whereas stereochemically-identical lesions in other sequence contexts were often poor substrates for replication. Similarly, R- and S-SO adducts introduced within identical sequences were often bypassed nonequivalently. Significantly, the degree of adduct-directed termination and translesion synthesis during replication was also dependent on the choice of polymerase. Although SO adducts directed termination either opposite the lesion or 1 base 3' to the damage using all five polymerases, templates that were poor substrates for bypass synthesis with one enzyme were often read-through much more efficiently when a different polymerase was used. Thus, the activities of these enzymes on the SO-modified substrates produced replication profiles, or "fingerprints", that were unique to each polymerase.
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PMID:The efficiency of translesion synthesis past single styrene oxide DNA adducts in vitro is polymerase-specific. 757 29

The ability of Klenow polymerase I, phage T7 polymerase (Sequenase), human polymerase alpha, and human polymerase beta to synthesize past (bypass) O6-methylguanine (O6-meG) lesions was studied in the presence of MgCl2 and MnCl2. An end-labeled 16-mer primer was annealed to the 3' end of gel-purified oligodeoxyribonucleotide templates (45-mers), each containing a single O6-meG in place of one G in the sequence -G1G2CG3G4T-. Extension products were analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. A fraction of the products extended by Klenow fragment terminated either opposite or one base before O6-meG located at sites 1 and 3. Termination occurred primarily one base before O6-meG located at sites 2 and 4. The remaining fractions that bypassed the lesions represented full-length product. In control reactions, the O6-meG-containing templates were annealed with complementary 45-mers, repaired with O6-alkylguanine DNA-alkyltransferase, annealed with an excess of labeled primer, and extended by Klenow fragment. Full-length extension of > 90% was observed with each template. Primer extension past O6-meG by DNA polymerase alpha and Sequenase was partially blocked in a manner which varied with the site of O6-meG in the template while primer extension by DNA polymerase beta was completely blocked (< 2% full length extension) with O6-meG at sites 1-4. Substitution of MnCl2 for MgCl2 in the reaction mixture greatly increased the bypass of O6-meG by Klenow fragment and DNA polymerase alpha but not Sequenase or DNA polymerase beta. The increased ability of Klenow fragment to bypass O6-meG in the presence of MnCl2 was found to result from an increased incorporation of G (O6-meG at sites 1 and 2) and A (O6-meG at sites 1, 2, and 3) opposite the lesion. The results indicate that O6-meG can block in vitro polymerization by several DNA polymerases and are consistent with the observed cytotoxic effects of methylating agents on mammalian cells.
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PMID:O6-methylguanine-induced replication blocks. 763 3

The products from base-specific, dideoxy-nucleotide chain-termination DNA sequencing reactions catalyzed by the modified T7 DNA polymerase have been analyzed by using the technique of matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Preliminary experiments were performed to determine detection limits for a synthetic mixture of mixed-base single-stranded DNA which contained a 14-mer, a 21-mer, and a 41-mer; acceptable spectra, showing peaks for each component, were obtainable for samples that contained as little as 5 fmol per component. Initial sequencing reactions were therefore carried out on 2-pmol amounts of a short synthetic template that was 45 nucleotides in length, employing 2 pmol of 12-mer as the primer strand. This provided readable sequence information out to the 19th base past the primer. Using a 21-mer primer, nearly the entire sequence of the template could be read.
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PMID:Analysis of enzymatic DNA sequencing reactions by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. 767 Jan 56

Homogeneously purified DNA polymerase alpha subunit-free primase was used to analyze primer RNA synthesis. On a chemically synthesized 36 mer DNA template, a part of upstream region of human c-myc gene, the primer synthesis started from a doublet of deoxythymidine (TT) in the deoxypyrimidine-rich sequence. The primase in DNA polymerase alpha-primase complex synthesized 21-mer reaction product, while DNA polymerase alpha-free primase gave the similar products, 21- and 22-mer, indicating that the site recognition was carried out by primase itself and DNA polymerase alpha subunit has an auxiliary role on it. Product analysis using DNA fragments carrying base substitutions further revealed that the existence of deoxypyrimidine residues around the starting sites was important for priming frequencies. Competition analysis showed that the priming was strongly competed by poly(dC), and to a much lesser extent by poly(dA). Gel-shift analysis showed that the primase could bind to the DNA template, and this complex formation was also competed by poly(dC), but not by poly(dA). These results indicate that primase subunit interacts with the starting site by binding directly with deoxypyrimidine residues.
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PMID:Deoxypyrimidine cluster mediates the priming by calf thymus DNA primase subunit. 768 46

Irradiation of d(GTATTATG) with 254 nm light gave rise to four major photoproducts, two of which were readily identified by NMR as the cis-syn cyclobutane dimer and the (6-4) photoproduct of the central TT site. Analysis of the NMR data for the other two photoproducts indicated that they were not any of the other known photoproducts of a TT site and might be TA* photoproducts [Bose, S. N., et al. (1983) Science 220, 723-725]. In support of this possibility, the fluorescence spectra of the products of acid hydrolysis of the two photoproducts were very similar to that reported for the hydrolysis product of the TA* photoproduct of TpdA. Only one of the two TA*-containing octamers could be ligated at both ends to form a 49-mer oligonucleotide in the presence of a complementary oligonucleotide scaffold, suggesting that the TA* photoproduct had formed between T5 and A6. The position of the TA* photoproduct was confirmed by mapping the arrest sites for 3'-->5' exonucleolytic degradation of the 49-mer by T4 DNA polymerase and for primer extension opposite the 49-mer by exonuclease deficient Klenow fragment (KF) and Sequenase Version 2.0. The TA* product could also be bypassed by both polymerases, but it was less of a block to KF. Treatment with 1 M aqueous piperidine at 100 degrees C led to a maximum of about 34% cleavage of the DNA at the site of the TA* product.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Preparation and characterization of a deoxyoligonucleotide 49-mer containing a site-specific thymidylyl-(3',5')-deoxyadenosine photoproduct. 782 86


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