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 synthetic procedure has been developed by which stable abasic sites are introduced into oligodeoxynucleotides at any desired position in the sequence. A modified tetrahydrofuran moiety, isosteric with 2'-deoxyribofuranose, serves as a structural analog of the natural apurinic/apyrimidinic site. We have also prepared oligodeoxynucleotides that lack cyclic structure at the abasic site but retain the carbon atoms of the phosphodiester backbone. These synthetic oligodeoxynucleotides are cleaved on the 5' side of the abasic site by endonuclease IV and by exonuclease III; they serve also as templates for avian myeloblastosis virus reverse transcriptase, Escherichia coli DNA polymerase I (Klenow fragment), and calf thymus DNA polymerase-alpha. Extension of primed templates by these DNA polymerases is blocked initially at the position immediately 3' to the abasic site; nucleoside monophosphates are subsequently incorporated opposite the lesion. The nucleotide most frequently incorporated opposite all abasic sites, regardless of structure, is dAMP. Significant "readthrough" at the abasic site was observed in experiments using avian myeloblastosis virus reverse transcriptase and DNA polymerase-alpha and, to a much lesser degree, with DNA polymerase I. We conclude that a modified tetrahydrofuran group can serve as a stable structural analog of 2'-deoxyribose in the apurinic/apyrimidinic site. These modified oligodeoxynucleotides should prove useful for studies of chemical mutagenesis.
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PMID:Oligodeoxynucleotides containing synthetic abasic sites. Model substrates for DNA polymerases and apurinic/apyrimidinic endonucleases. 244 Aug 61

Human DNA polymerase alpha holoenzyme follows an ordered sequential terreactant mechanism of substrate recognition and binding (Wong, S. W., Paborsky, L. R., Fisher, P. A., Wang, T. S.-F., and Korn, D. (1986) J. Biol. Chem. 261, 7958-7968). We confirmed this mechanism for the DNA polymerase alpha holoenzyme purified from Drosophila melanogaster embryos and studied the interaction of Drosophila pol alpha with synthetic oligonucleotide template-primers containing modified tetrahydrofuran moieties as model abasic lesions chemically engineered at a number of defined sites. Abasic lesions in the template had relatively little effect on the polymerase incorporation reaction at sites proximal to the lesion. However, incorporation opposite an abasic site was undetectable relative to that which occurred opposite a normal template nucleotide. Moreover, abasic residues in the primer region of the template-primer construct as far as 4 base pairs removed from the 3'-primer terminus prevented detectable nucleotide incorporation relative to that seen on an unmodified template-primer. Primer-region lesions had qualitatively similar effects whether they were located on the primer strand itself or on the complementary template strand. Data from polymerase incorporation experiments were corroborated by competitive binding assays performed under steady state reaction conditions. Results of these experiments suggested that polymerase binding to synthetic oligonucleotide template-primers was essentially unaffected by lesions located at sites that did not block incorporation. Lesions that did block incorporation apparently did so by abrogating template-primer binding. These observations have implications for understanding the mechanisms whereby DNA polymerase alpha recognizes noninformational template sites in vivo and prevents DNA synthesis from proceeding past these points.
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PMID:Recognition and binding of template-primers containing defined abasic sites by Drosophila DNA polymerase alpha holoenzyme. 250 45

The activities of 5-methyltetrahydrofolate (5-CH3-THF)-related enzymes [5-CH3-THF homocysteine methyltransferase and 5,10-methylenetetrahydrofolate (5,10-CH2-THF) reductase] and DNA polymerase alpha were measured in normal and malignant hematopoietic cells. The 5-CH3-THF homocysteine methyltransferase activity was significantly correlated with 5,10-CH2-THF reductase activity, indicating that the hematopoietic cells with active biosynthesis of tetrahydrofolate from 5-CH3-THF also actively synthesize 5-CH3-THF from 5,10-CH2-THF. The activities of 5-CH3-THF-related enzymes had a tendency to be high in lymphoid cells and low in myeloid cells, and were not correlated with the percentage of blasts and immature cells in the samples examined. Fairly good correlations were observed among these three enzymes in non-malignant bone marrow cells. However, the activities of two of the enzymes correlated only weakly overall with DNA polymerase alpha activity in normal and malignant hematopoietic cells. Generally speaking, DNA polymerase alpha activity correlated well with the percentage of blasts and immature cells in the samples examined.
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PMID:5-methyltetrahydrofolate-related enzymes and DNA polymerase alpha in normal and malignant hematopoietic cells. 635 15

Mitochondrial DNA is subject to oxidative damage generating 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) residues and to spontaneous or induced base loss generating abasic sites. Synthetic oligonucleotides containing these lesions were prepared and used as templates to determine their effects on the action of Xenopus laevis DNA polymerase gamma. An analogue of an abasic site in DNA, tetrahydrofuran, was found to inhibit elongation by DNA polymerase gamma. When the DNA polymerase was able to complete translesional synthesis, a dA residue was incorporated opposite the abasic site. In contrast, elongation by DNA polymerase gamma was not inhibited by an 8-oxo-dG residue in the template strand. The polymerase inserted dA opposite 8-oxo-dG in approximately 27% of the extended products. The effects of these lesions on the 3'-->5' exonuclease proofreading activity of DNA polymerase gamma were also investigated. The 3'-->5' exonuclease activity excised any of the four normal bases positioned opposite either a tetrahydrofuran residue or 8-oxo-dG, suggesting that proofreading may not play a major role in avoiding misincorporation at abasic sites or 8-oxo-dG residues in the template. Thus, both of these lesions have the prospect of causing high rates of mutation during mtDNA replication.
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PMID:Action of mitochondrial DNA polymerase gamma at sites of base loss or oxidative damage. 772 37

DNA damage frequently leads to the production of apurinic/apyrimidinic (AP) sites, which are presumed to be repaired through the base excision pathway. For detailed analyses of this repair mechanism, a synthetic analog of an AP site, 3-hydroxy-2-hydroxymethyltetrahydrofuran (tetrahydrofuran), has been employed in a model system. Tetrahydrofuran residues are efficiently repaired in a Xenopus laevis oocyte extract in which most repair events involve ATP-dependent incorporation of no more than four nucleotides (Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 9:3750-3757, 1989; Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 11:4441-4447, 1991). Using a series of column chromatography procedures to fractionate X. laevis ovarian extracts, we developed a reconstituted system of tetrahydrofuran repair with five fractions, three of which were purified to near homogeneity: proliferating cell nuclear antigen (PCNA), AP endonuclease, and DNA polymerase delta. This PCNA-dependent system repaired natural AP sites as well as tetrahydrofuran residues. DNA polymerase beta was able to replace DNA polymerase delta only for repair of natural AP sites in a reaction that did not require PCNA. DNA polymerase alpha did not support repair of either type of AP site. This result indicates that AP sites can be repaired by two distinct pathways, the PCNA-dependent pathway and the DNA polymerase beta-dependent pathway.
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PMID:Proliferating cell nuclear antigen-dependent abasic site repair in Xenopus laevis oocytes: an alternative pathway of base excision DNA repair. 791 6

Site-specifically modified oligodeoxynucleotides containing a single natural abasic site or a chemically synthesized (tetrahydrofuran or deoxyribitol) model abasic site were used as templates for primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I or by calf thymus DNA polymerase alpha. Analysis of the fully extended products of these reactions indicated that both polymerases preferentially incorporate dAMP opposite the natural abasic site and tetrahydrofuran, while DNA templates containing the ring-opened deoxyribitol moiety block translesional synthesis, promoting sequence context-dependent deletions. The frequency of nucleotide insertion opposite the three types of abasic sites follows the order dAMP > dGMP > dCMP > dTMP. The frequency of chain extension was highest when dAMP was positioned opposite a natural abasic site. The frequency of translesional synthesis past abasic sites follows the order tetrahydrofuran > deoxyribose > deoxyribitol. The Klenow fragment promotes blunt end addition of dAMP; this reaction was much less efficient than insertion of dAMP opposite an abasic site. We conclude that the miscoding potential of a natural abasic site in vitro closely resembles that of its tetrahydrofuran analog. Ring-opened abasic sites favor deletions. Studies with polymerase alpha in vitro predict preferential incorporation of dAMP at abasic sites in mammalian cells.
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PMID:Translesional synthesis on DNA templates containing a single abasic site. A mechanistic study of the "A rule". 915 53

Mutagenic DNA adducts have been analyzed with respect to the rate of nucleotide insertion opposite the modified base, extension from that "mispair", and nucleotide insertion preference. To complement and extend these studies we have investigated the long-range effects of DNA adducts on DNA polymerase activity. To address this question, primer extension reactions were performed using DNA polymerase I, Klenow fragment exo-. Templates containing 7,8-dihydro-8-oxoguanine, dG-C8-aminofluorene, dG-C8-(acetylamino)fluorene, and the model abasic site, tetrahydrofuran, were used for these studies, and the steady-state kinetics of correct nucleotide insertion were determined at positions (-2), (-1), (+1), (+2), (+3), and (+5) with respect to the template lesion. The kinetics of primer extension by Klenow fragment exo- at template positions 3' to the lesion showed only a small inhibitory effect, <3-fold, even for the strongly blocking lesion, dG-C8-(acetylamino)fluorene, indicating that Klenow fragment exo- activity is not greatly affected by lesions in the single-stranded portion of the template-primer. In contrast, a dramatic decrease in the frequency of primer extension was observed at template sites 5' to the site of adduction. Inhibition of polymerase activity decreased as the distance from the lesion increased; however, a relatively large effect was seen at the (+2) and (+3) positions for dG-C8-(acetylamino)fluorene and tetrahydrofuran. For these blocking lesions, the effect on extension 5 bases from the lesion was greatly reduced. We conclude from these studies that DNA damage at positions remote from the site of the lesion affects DNA polymerase function.
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PMID:Kinetics of DNA polymerase I (Klenow fragment exo-) activity on damaged DNA templates: effect of proximal and distal template damage on DNA synthesis. 939 62

The influence of sequence context on the ability of DNA polymerase to bypass sites of base loss was addressed using an in vitro selection system. Oligonucleotides containing either an aldehydic abasic site or tetrahydrofuran surrounded by four randomized bases on both the 5' and 3' sides were used as templates for synthesis by phage T4 DNA polymerase holoenzyme proficient or deficient in the 3'-->5' proofreading exonuclease activity. Successful bypass products were purified, subcloned and the sequences of approximately 100 subclones were determined for each of the four polymerase/lesion combinations tested. Between 7 and 19 % of the bypass products contained deletions of one to three nucleotides in the randomized region. In bypass products not containing deletions, biases for and against certain nucleotides were readily noticeable across the entire randomized region. Template strands from successful bypass products of abasic sites had a high frequency of T in most of the randomized positions, while those from bypass products of tetrahydrofuran had a high frequency of G at the positions immediately to the 3' and 5' side of the lesion. Consensus sequences were shared by successful bypass products of the same lesion but not between bypass products of the two lesions. The consensus sequence for efficient bypass of tetrahydrofuran was over-represented in several frames relative to the lesion. T4 DNA polymerase inserted A opposite abasic sites 63 % of the time in the presence of proofreading and 79 % of the time in its absence, followed by G>T>C, while the insertion of A opposite tetrahydrofuran ranged between 93 % and 100 % in the presence and absence of proofreading, respectively. Finally, sequence context influenced the choice of nucleotide inserted opposite abasic sites and consensus sequences which favored the incorporation of nucleotides other than A were defined.
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PMID:In vitro selection of sequence contexts which enhance bypass of abasic sites and tetrahydrofuran by T4 DNA polymerase holoenzyme. 1004 81

Cidofovir is the first nucleoside monophosphate analogue currently being used for the treatment of human cytomegalovirus (HCMV) retinitis in individuals with AIDS. Unfortunately, the period of therapy with the use of this compound may be limited due to the possible emergence of serious irreversible nephrotoxic effects. New drugs with improved toxicity profiles are needed. The goal of this study was to investigate the anticytomegaloviral properties and drug-induced toxicity of a novel phosphonate analogue, namely, (-)-2-(R)-dihydroxyphosphinoyl-5-(S)-(guanin-9'-yl-methyl) tetrahydrofuran (compound 1), in comparison with those of cidofovir. The inhibitory activities of both compounds on HCMV propagation in vitro were similar against the AD 169 and Towne strains, with 50% inhibitory concentrations ranging from 0.02 to 0.17 microgram/ml for cidofovir and < 0.05 to 0.09 microgram/ml for compound 1. A clinical HCMV isolate that was resistant to ganciclovir and that had a known mutation within the UL54 DNA polymerase gene and a cidofovir-resistant laboratory strain derived from strain AD 169 remained sensitive to compound 1, whereas their susceptibilities to ganciclovir and cidofovir were reduced by 33- and 10-fold, respectively. Both compound 1 and cidofovir exhibited equal potencies in an experimentally induced murine cytomegalovirus (MCMV) infection in mice, with a prevention or prolongation of mean day to death at dosages of 1.0, 3.2, and 10.0 mg/kg of body weight/day. In cytotoxicity experiments, compound 1 was found to be generally more toxic than cidofovir in cell lines Hs68, HFF, and 3T3-L1 (which are permissive for HCMV or MCMV replication) but less toxic than cidofovir in MRC-5 cells (which are permissive for HCMV replication). Drug-induced toxic side effects were noticed for both compounds in rats and guinea pigs in a 5-day repeated-dose study. In guinea pigs, a greater weight loss was noticed with cidofovir than with compound 1 at dosages of 3.0 and 10.0 mg/kg/day. An opposite effect was detected in rats, which were treated with the compounds at relatively high dosages (up to 100 mg/kg/day). Compound 1 and cidofovir were nephrotoxic in both rats and guinea pigs, with the epithelium lining the proximal convoluted tubules in the renal cortex being the primary target site. The incidence and the severity of the lesions were found to be dose dependent. The lesions observed were characterized by cytoplasm degeneration and nuclear modifications such as karyomegaly, the presence of pseudoinclusions, apoptosis, and degenerative changes. In the guinea pig model, a greater incidence and severity of lesions were observed for cidofovir than for compound 1 (P < 0.001) with a drug regimen of 10 mg/kg/day.
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PMID:Comparative study of the anti-human cytomegalovirus activities and toxicities of a tetrahydrofuran phosphonate analogue of guanosine and cidofovir. 1004 67

We recently proposed a mechanism for why dAMP is primarily inserted opposite both T's of photoproducts of TT sites by T7 DNA polymerase [Smith, C. A., Baeten, J., and Taylor, J.-S. (1998) J. Biol. Chem., 273, 21933-21940] that was based on analysis of a recent crystal structure of a complex of this enzyme with a template, a primer, and a dideoxynucleotide. We proposed that indiscriminate insertion of dAMP opposite the 3'-T of each photoproducts takes place via a transient abasic site-like intermediate, with the photoproduct outside the active site, whereas insertion of dAMP opposite the 5'-T takes place with the photoproduct inside the active site. To obtain further support for this mechanism, we have investigated the selectivity of dNMP and pyrene nucleotide (dPMP) insertion opposite each T of the cis,syn, trans,syn-I, trans,syn-II, (6-4), and Dewar photoproducts of TT and opposite a tetrahydrofuran abasic site analogue by the exonuclease-deficient T7 DNA polymerase, Sequenase Version 2.0. Selectivity was determined by a direct competition assay that makes use of a stacked gel to resolve the various extension products. Pyrene nucleotide was chosen for investigation because it has been previously shown to be selectively inserted opposite abasic sites and was therefore expected to probe whether the photoproducts were inside the active site during a particular insertion step. In accord with the proposed mechanism, dPMP was inserted in preference to dAMP opposite the 3'-T of all the photoproducts with the exception of the trans,syn-I product, whereas dAMP was inserted in preference to dPMP opposite the 5'-T of all the photoproducts. In addition to supporting the proposed mechanism, these results suggest that pyrene nucleotide may be a useful probe for investigating the mechanism of DNA damage bypass by polymerases and for characterizing their active sites.
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PMID:Pyrene nucleotide as a mechanistic probe: evidence for a transient abasic site-like intermediate in the bypass of dipyrimidine photoproducts by T7 DNA polymerase. 1108 16


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