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 effect of metal activators on the fidelity of DNA synthesis has been examined. Using the DNA polymerase from avian myeloblastosis virus, the accuracy of Co2+-, M2+-, and Ni2+-activated DNA synthesis was determined with different polynucleotide templates. With poly[d(A-T)] as the template, the error frequency for dCMP incorporation was 1:1400, 1:1100, and 1:600 for Mg2+, Co2+, and Mn2+, respectively, at maximally activating concentrations. The error frequency was invariant with respect to [Mg2+] but increased with greater than activating concentrations of Co2+ and Mn2+. This increase resulted from differential rates of complementary and noncomplementary nucleotide incorporation. The enhanced error frequency was nonspecific as it occurred with all polynucleotide templates and with all noncomplementary deoxy- and ribonucleotides which were tested. Nearest neighbor analyses of the reaction products indicated that the noncomplementary deoxynucleotides were incorporated as single base substitutions. The fidelity of Ni2+-activated DNA synthesis was invariant with respect to [Ni2+] and was similar to that obtained using Mg2+. During DNA synthesis with Mg2+, the addition of Co2+, Mn2+, or Ni2+ resulted in a decrease in the fidelity of DNA synthesis. The relationship between decreases in the fidelity of DNA synthesis and metal mutagenesis, or carcinogenesis, or both, is considered.
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PMID:On the fidelity of DNA replication. Effect of metal activators during synthesis with avian myeloblastosis virus DNA polymerase. 86 97

On the basis of chemical considerations and model building, the Watson-Crick concept of complementary base pairing is extended to a wider range of DNA pairs that A-T and G-C (including A-C, G-T, A-A, G-G and G-A) by invoking imino or enol tautomers (or protonated species) and synisomers. The virtual absence of these additional base pairs from DNA is explained in terms of the low frequency with which these unfavoured forms occur and the two-step mechanism of DNA synthesis, whereby residues are first incorporated by the DNA polymerase and then checked. This base-pairing hypothesis is used to explain the origin, nature and level of spontaneous substitution mutations, their enhancement by base analogues, and the unique effects of certain mutator alleles.
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PMID:Complementary base pairing and the origin of substitution mutations. 95 82

A DNA-nuclear membrane complex has been isolated by two different methods from the nuclei of cultured mouse fibroblast (3T3) cells. One method, utilizing the detergent sarkosyl (sodium lauroyl sarkosinate), yields a DNA-nuclear membrane complex (the M band), which contains virtually all of the DNA in the nuclei. However, treatment of the M band by sonication, vortexing, or freeze-thaw reduces the amount of DNA in the complex by approximately 50-80%, depending upon the phase of the cell cycle from which the complex was extracted. The remaining DNA is tightly bound to the nuclear membrane and resists further shearing procedures. Over 90% of the choline-labeled phospholipid present in nuclei is also found in these sheared M bands. The percentage of DNA associated with the nuclear membrane varies during the cell cycle and correlates well with the onset, continuation, and cessation of DNA synthesis. Thus, although DNA-membrane complexes can be detected throughout the cell cycle, the percentage of DNA bound to membrane increases during late G1 and S and decreases during G2. In addition, there are distinct qualitative differences in the type of DNA present in the membrane fraction, with a more highly d(A-T) rich DNA being present in confluent (G0) cells than in cells during the S phase. This d(A-T) rich DNA may be related to the mouse satellite DNA identified by others. The M band can be separated into two DNA-nuclear membrane subfractions by centrifugation through a continuous sucrose gradient. The relative proportions of these two subfractions depend upon the percentage of sarkosyl present in the M band prior to centrifugation, with complete removal of sarkosyl resulting in a very large increase in the sedimentation velocity of the complex and in the formation of only one fraction. Evidence that this is a complex of DNA with membrane is given by the finding that DNA is dissociated from the complex with Pronase, deoxycholate, or high levels of sarkosyl. Removal of virtually all of the DNA with DNase from this rapidly sedimenting complex does not dissociate any of the phospholipid which still sediments rapidly as a single band. A second method, which yields a DNA-membrane fraction from nuclei, utilizes sedimentation of lysed nuclei to equilibrium in CsCl density gradients. This low-density CsCl fraction contains only 10-15% of the total DNA, but contains most of the nascent DNA, which may be chased into a membrane-free fraction. The DNA-membrane fraction from CsCl gradients possesses properties in common with the M-band fraction and can be converted into an M band. DNA membrane complexes from sucrose gradients, as well as the crude M-band preparation and a non-membrane-associated DNA fraction from nuclei can synthesize DNA in vitro without the addition of an external DNA template or DNA polymerase. In contrast to the activity in the non-membrane-associated DNA fraction, the membrane-associated polymerase activity is strongly stimulated by adenosine triphosphate and is unaffected by ethidium bromide...
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PMID:A nuclear membrane-associated DNA complex in cultured mammalian cells capable of synthesizing DNA in vitro. 99 Feb 45

The antibiotic steffimycin B binds to double-stranded DNA as evidenced by difference spectroscopy and an increase of the thermal stability of DNA in the presence of the antibiotic. Salmon sperm DNA-steffimycin B complexes show a drastic decrease in template activity for Escherichia coli DNA polymerase I but not for DNA-idrected RNA polymerase. The differences in template properties of poly[d(A-T)] and poly (dG) - poly(dC)-antibiotic complexes,respectively, for DNA polymerase I and RNA polymerase suggest that the antibiotic interacts primarily with adenine or thymine bases or both in double-stranded DNA.
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PMID:Steffimycin B, a DNA binding agent. 109 Mar 4

A steady state kinetic study of Escherichia coli DNA polymerase I has been carried out using poly[d(A-T)] as the template-primer substrate. The results of substrate saturation and product inhibition kinetic studies suggest an altered Ordered Bi Bi mechanism for the enzyme. The Michaelis constants for polymer, d-atp, and dTTP are 5 nM (3'-OH ends), 1 muM, and 2 muM, respectively. The apparent equilibrium constant for the reaction, Keq equals [PPi]/[dNTP], was estimated as greater than or equal to 500. No quaternary complex of enzyme, template, and both deoxynucleoside triphosphates was detected. Single turnover experiments at 4 degrees indicated that the enzyme functions non-processively under the specified conditions, that is, dissociates after each catalytic step. The results at higher temperature were consistent with dissociation within 30 steps. Furthermore, at 4 degrees a burst of incorporation stoichiometric with the amount of enzyme was observed upon initiation of the reaction, indicating that the rate-limiting step in the steady state occurs after phosphodiester bond formation. There is a linear Arrhenius dependence of the initial reaction on temperature in the range 4-40 degrees, with an apparent Ea equals 17 kcal/mol. The rate equations appropriate for template-dependent polymerases which dissociate after each catalytic step have been derived.
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PMID:The steady state kinetic parameters and non-processivity of Escherichia coli deoxyribonucleic acid polymerase I. 109 83

DNA primases encoded by the conjugative plasmids ColIb-P9 (IncI1), RP4, and R751 (IncP), and the protein of the Escherichia coli satellite phage P4 alpha were shown to contain a common amino acid sequence motif -E-G-Y-A-T-A-. The P4 alpha gene product, required for initiation of phage DNA replication, exhibits primase activity on single-stranded circular DNA templates. This priming activity resembles the enzymatic activity of DNA primases encoded by conjugative plasmids in terms of template utilization and the ability to synthesize primers that can be elongated by DNA polymerase III holoenzyme. The -E-G-Y-A-T-A- motif is part of an extended sequence region most conserved within the primase domains of the four enzymes. Single amino acid substitutions generated in the -E-G-Y-A-T-A- motif of the RP4 TraC2 and the P4 alpha protein affect priming activity, supporting the hypothesis that the conserved sequence motif is part of the active center for primase function. A mutation that eliminates priming activity causes P4 phage to grow poorly and to depend upon the host dnaG primase. Computer analysis identified two additional sequence motifs within the amino acid sequence of the P4 alpha protein: a potential zinc-finger motif and a "type A" nucleotide binding site, both strikingly similar to sequence motifs described in various DNA primases and helicases.
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PMID:A common sequence motif, -E-G-Y-A-T-A-, identified within the primase domains of plasmid-encoded I- and P-type DNA primases and the alpha protein of the Escherichia coli satellite phage P4. 161 4

CC-1065, a cyclopropylpyrroloindole (CPI), is a highly potent antitumor DNA-alkylating agent. We have devised a simple method to detect CPI bonding sites on double-stranded DNA (dsDNA). The technique utilizes a modified form of bacteriophage T7 polymerase, Sequenase, to synthesize a radiolabeled nascent strand from dsDNA that has been reacted in vitro with the CC-1065 analogue U-73975 (adozelesin). The reaction products were electrophoresed on sequencing gels containing 8 M urea and visualized by autoradiography. The transit of this DNA polymerase is inhibited at the sites where CPIs are bound to the template strand. Thus, the enzyme stalls or stops at the nucleotide immediately adjacent to the modified base, resulting in the accumulation of DNA strands at these sites and in diminished read-through beyond these sites in a set of CPI-treated DNA molecules. The precise positions of polymerase inhibition can be determined by comparison of CPI-treated and unreacted DNA reactions. This modified dideoxynucleotide sequencing technique has been used to establish the sequence selectivity of U-73975. Approximately 1 kilobase of dsDNA has been analyzed to derive a consensus canonical bonding sequence, 5'(T/A)-T/A-T-A*-(C/G)-(G), where A* is the site of U-73975 alkylation and parentheses denote deoxynucleotide preferences. Noncanonical sites were also found at poly(A) sites. This technique yielded a consensus sequence for U-73975 bonding that is similar to, but not identical with, the published consensus obtained for CC-1065 by a modified Maxam and Gilbert sequencing technique. We have also examined the bonding of [3H]U-73975 to the DNA of viable cultured mammalian cells, using gel electrophoresis and autoradiographic techniques.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:In vitro and in vivo DNA bonding by the CC-1065 analogue U-73975. 185 54

The DNA and protein sequences of single-stranded DNA binding proteins (SSBs) encoded by the plP71a, plP231a, and R64 conjugative plasmids have been determined and compared to Escherichia coli SSB and the SSB encoded by F-plasmid. Although the amino acid sequences of all of these proteins are highly conserved within the NH2-terminal two-thirds of the protein, they diverge in the COOH-terminal third region. A number of amino acid residues which have previously been implicated as being either directly or indirectly involved in DNA binding are conserved in all of these SSBs. These residues include Trp-40, Trp-54, Trp-88, His-55, and Phe-60. On the basis of these sequence comparisons and DNA binding studies, a role for Tyr-70 in DNA binding is suggested for the first time. Although the COOH-terminal third of these proteins diverges more than their NH2-terminal regions, the COOH-terminal five amino acid residues of all five of these proteins are identical. In addition, all of these proteins share the characteristic property of having a protease resistant, NH2-terminal core and an acidic COOH-terminal region. Despite the high degree of sequence homology among the plasmid SSB proteins, the F-plasmid SSB appears unique in that it was the only SSB tested that neither bound well to poly(dA) nor was able to stimulate DNA polymerase III holoenzyme elongation rates. Poly [d(A-T)] melting studies suggest that at least three of the plasmid encoded SSBs are better helix-destabilizing proteins than is the E. coli SSB protein.
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PMID:Single-stranded DNA binding proteins (SSBs) from prokaryotic transmissible plasmids. 200 32

Mutations induced in cultured human cells by 254-nm UV light were analyzed within exon 3 of the hypoxanthine guanine phosphoribosyl transferase (HPRT) gene. Five large independent cultures of human lymphoblastoid cells, line TK6, were exposed to 4 J/m2 of 254-nm UV light and mutants at the HPRT locus were selected en masse by 6-thioguanine (6TG) resistance. Exon 3 of the HPRT gene was amplified from the mutant cells by polymerase chain reaction (PCR) using modified T7 DNA polymerase. Denaturing gradient gel electrophoresis (DGGE) was used to separate the mutant sequences from the wild type as mutant/wild-type heteroduplexes. Individual mutant bands were isolated from the gel and the nature of the mutations was determined by direct sequencing. Eight predominant mutations were detected in the 184-bp exon 3 sequence. Of these, 3 transition, including 2 G-C to A-T and 1 A-T to G-C and 2 A-T to C-G transversions, appeared in all 5 UV-treated cultures but not in untreated cultures and were thus considered to be mutational hotspots. These observations are similar in nature to those previously reported in bacterial and rodent cells. A single G deletion, a tandem substitution of CpT for TpA, and a tandem triple substitution of GpGpA for ApApG were also observed but in only 2, 2 and 3 of the 5 UV-treated cultures, respectively. Numerical analysis of the mutant fractions of these 8 mutations indicated that each of them was distributed as a set of non-random and independent events, i.e., a mutational hotspot.
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PMID:Analysis of point mutations induced by ultraviolet light in human cells. 206 29

The rat mitochondrial single strand DNA binding protein (SSB) P16 was purified to apparent homogeneity by elution from single strand DNA agarose with ethidium bromide. Each monomer of P16 contains two tryptophan residues, and the intrinsic fluorescence from these residues is quenched upon binding to single strand polynucleotides. From fluorescence quench titrations of ligand to fixed amounts of DNA lattice, a binding site size of 8 or 9 nucleotides per P16 monomer was found. Measurement of the affinity of P16 for isolated sites by titration with either oligo(dT)8 or 5'-dephosphorylated oligo(dT)8 indicated values on the order of 10(7) M-1. P16 exhibited a binding preference for single strand DNA, poly(dT), and poly(dC) in comparison to double strand DNA, poly(U), or poly[d(A-T)]. Although it was not possible to show that P16 destabilizes double helical DNA or even poly[d(A-T)], binding of P16 does inhibit the process of renaturation as shown by inhibition of duplex formation between poly(dA) and poly(dT). The binding of saturating amounts of P16 to single strand poly(dT).oligo(dA)50 template-primers enhanced approximately 10-fold the activity of both the homologous mitochondrial DNA polymerase and the Escherichia coli DNA polymerase I Klenow fragment. However, the mitochondrial DNA primase was nearly completely inhibited by the saturation of the poly(dT) template with P16. Amino-terminal sequence analysis of P16 and a protease-insensitive, DNA binding domain (Mr approximately 6000) revealed that the DNA binding domain residues, at least in part, in the amino-terminal third of the P16 molecule. Furthermore, the amino-terminal sequence was found to be strikingly similar to that of the Xenopus laevis mtSSB-1 and to a lesser extent similar to E. coli SSB and E. coli F sex factor SSB.
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PMID:Structural and functional studies of the rat mitochondrial single strand DNA binding protein P16. 222 14


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