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 pcbA1 mutation allows DNA replication dependent on DNA polymerase I at the restrictive temperature in polC(Ts) strains. Cells which carry pcbA1, a functional DNA polymerase I, and a temperature-sensitive DNA polymerase III gene were used to study the role of DNA polymerase III in DNA repair. At the restrictive temperature for DNA polymerase III, these strains were more sensitive to the alkylating agent methyl methanesulfonate (MMS) and hydrogen peroxide than normal cells. The same strains showed no increase in sensitivity to bleomycin, UV light, or psoralen at the restrictive temperature. The sensitivity of these strains to MMS and hydrogen peroxide was not due to the pcbAl allele, and normal sensitivity was restored by the introduction of a chromosomal or cloned DNA polymerase III gene, verifying that the sensitivity was due to loss of DNA polymerase III alpha-subunit activity. A functional DNA polymerase III is required for the reformation of high-molecular-weight DNA after treatment of cells with MMS or hydrogen peroxide, as demonstrated by alkaline sucrose sedimentation results. Thus, it appears that a functional DNA polymerase III is required for the optimal repair of DNA damage by MMS or hydrogen peroxide.
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PMID:DNA polymerase III requirement for repair of DNA damage caused by methyl methanesulfonate and hydrogen peroxide. 244 82

9-beta-D-Arabinofuranosyladenine (ara-A) is an inhibitor of DNA replication with antitumor and antiviral activity. The molecular basis for this inhibitory effect has remained unclear. The present work has examined the effects of 9-beta-D-arabinofuranosyladenine-triphosphate on DNA polymerase-beta. These studies were performed on different M13 phage DNA templates. The findings demonstrate that 9-beta-D-arabinofuranosyladenine is incorporated into the elongating DNA strand by DNA polymerase-beta. The findings also demonstrate that the incorporated 9-beta-D-arabinofuranosyladenine residue acts as a relative chain terminator. Furthermore, the relative chain-terminating effects of this agent are sequence specific and reversed by competition with deoxyadenosine-triphosphate for incorporation into the DNA strand. These findings are in concert with hydrogen bonding differences of the incorporated arabinosyl moiety which alters reactivity of the chain terminus and thereby inhibits elongation. These findings are also in agreement with recent studies of 1-beta-D-arabinofuranosylcytosine and provide insights into the sequence specific effects of these agents.
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PMID:Sequence-specific inhibition of DNA strand elongation by incorporation of 9-beta-D-arabinofuranosyladenine. 246 40

Binding of a small antisense RNA (RNA I) to the primer transcript (RNA II) of plasmid ColE1 inhibits formation of primer for DNA polymerase I-mediated plasmid replication. It is thought that RNA I and RNA II transiently interact via their single-stranded loop regions to form an unstable complex that subsequently converts into a more stable complex by hybridization. Rom (or Rop) protein enhances the inhibitory effect of RNA I on replication by enhancing the binding of the two RNAs. In this paper, we develop a model for the kinetics of the RNA I-RNA II binding reaction, estimate the rate constants, and provide a quantitative description of the effects of Rom protein. We show that the reaction kinetics are consistent with a stepwise binding model in which Rom protein binds to RNA I and RNA II, while the RNAs are held together in a transient complex. Mutations that replace C.G pairs by T.A pairs in the RNA loop regions and thus display weaker hydrogen bonding between the loop regions should be associated with an increased rate of dissociation for the unstable complex. Our model predicts that such destabilization of the loop interactions leads to a greater enhancement in the binding rate by Rom protein. The available data support this prediction.
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PMID:Kinetics of complementary RNA-RNA interaction involved in plasmid ColE1 copy number control. 247 39

A new method of estimation of dissociation constants for ligands and free energies of its binding based on the affinity modification of active centers in the presence of competitive ligands was developed. This method is designed for the analysis of protein-nucleic acid interactions in template systems. Deoxyoligoribonucleotides containing the reactive residue of cis-aquadihydroxydiaminoplatinum (II) and oligonucleotides ethylated at phosphate groups were used for the study of interactions of human placental DNA-polymerase alpha and the Klenow fragment of DNA-polymerase I from E. coli with templates and primers. A model was constructed which postulates the formation of a single Me2+-dependent electrostatic bond and of a hydrogen bond by one of template phosphates with the enzyme active center. Similar bonds form the basis for the enzyme interaction with the 3'-terminal phosphate group of the primer. Other monomeric units of the template are likely to interact with the enzyme by forming hydrophobic bonds. Other mononucleotide units of the primer are involved in complementary interactions with the template. The primer activity of dNMP and NMP in these systems has been demonstrated for the first time. The efficiency of dNMP, dNDP and dNTP interaction with DNA-polymerase was estimated from the affinity modification of the enzymes by dNTP and dNMP imidazolides. The key role of the template-primer interaction in the formation of the dNTP-binding site of DNA-polymerases was demonstrated. A significant contribution of dNTP gamma-phosphate to the template--dependent specific tuning of substrate dNTP was revealed.
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PMID:[Protein-nucleic acid interactions in reactions catalyzed by eukaryotic and prokaryotic DNA-polymerases]. 250 66

5-(p-Chlorobenzyl)-6-aminouracil (5-ClAU) inhibited RNA-dependent DNA polymerase (reverse transcriptase) from avian myeloblastosis virus. Inhibition was expressed only in the presence of a polyribonucleotide template such as mRNA or poly(rA), and kinetic analysis suggested that the action of 5-ClAU is competitive with template:primer. 5-ClAU did not inhibit HeLa DNA polymerase gamma, an enzyme that efficiently copies polyribonucleotide templates. A mechanism is proposed in which a 5-ClAU:template complex interferes with enzyme function, based partly on NMR studies indicating that 5-ClAU can form a hydrogen-bonded complex with deoxyadenosine in solution.
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PMID:Inhibition of RNA-directed DNA polymerase from avian myeloblastosis virus by a 5-benzyl-6-aminouracil. 257 88

The repair response of Escherichia coli to hydrogen peroxide has been examined in mutants which show increased sensitivity to this agent. Four mutants were found to show increased in vivo sensitivity to hydrogen peroxide compared with wild type. These mutants, in order of increasing sensitivity, were recA, polC, xthA, and polA. The polA mutants were the most sensitive, implying that DNA polymerase I is required for any repair of hydrogen peroxide damage. Measurement of repair synthesis after hydrogen peroxide treatment demonstrated normal levels for recA mutants, a small amount for xthA mutants, and none for polA mutants. This is consistent with exonuclease III being required for part of the repair synthesis seen, while DNA polymerase I is strictly required for all repair synthesis. Sedimentation analysis of cellular DNA after hydrogen peroxide treatment showed that reformation was absent in xthA, polA, and polC(Ts) strains but normal in a recA cell line. By use of a lambda phage carrying a recA-lacZ fusion, we found hydrogen peroxide does not induce the recA promoter. Our findings indicate two pathways of repair for hydrogen peroxide-induced DNA damage. One of these pathways would utilize exonuclease III, DNA polymerase III, and DNA polymerase I, while the other would be DNA polymerase I dependent. The RecA protein seems to have little or no direct function in either repair pathway.
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PMID:Multiple pathways for repair of hydrogen peroxide-induced DNA damage in Escherichia coli. 264 41

The modification of Klenow fragment of DNA polymerase I E. coli was investigated by the affinity reagents d(Tp)2C[Pt2+(NH3)2OH](pT)7 and d(pT)2pC[Pt2+(NH3)2OH](pT)7. The template binding site of the enzyme was modified by these reagents in the presence of NaF (5 mM), which inhibits selectively the 3'----5'-exonuclease activity of the enzyme and therefore prevents the reagent from degradation. NaCN destroyed covalent bonds between reagents and enzyme, restoring activity of the Klenow fragment. The affinity of different ligands (inorganic phosphate, nucleoside monophosphates, oligonucleotides) to the template binding site of Klenow fragment was estimated. Minimal ligands capable to bind with the template site were shown to be triethylphosphate (Kd 290 microM) and phosphate (Kd 26 microM). Ligand affinity increases by the factor 1.76 per an added (monomer unit from phosphate to d(pT) and then for oligonucleotides d(Tp)nT (n 1 to 19-20). At n greater than 19-20, the ligand affinity remained constant. The complete ethylation of phosphodiester groups lowers affinity of the oligothymidylates to the enzyme by approximately 10 times, and comparable decrease of Pt2+-oligonucleotide affinity to polymerase is caused by the absence of Mn2+-ions. The data obtained led to suggestion that one Me2+-dependent electrostatic contact of the template phosphodiester group with the enzyme takes place (delta G = -1.45...-1.75 kcal/mole). Formation of a hydrogen bond with the oxygen atom of P = O group of the same template phosphate is also assumed (delta G = -4.8...-4.9 kcal/mole). Other template internucleotide phosphates do not interact with the enzyme but the bases of oligonucleotides take part in hydrophobic interactions with the template binding site. Gibbs energy changes by -0.34 kcal/mole when the template is lengthened by one unit.
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PMID:[Klenow fragment of DNA-polymerase I from E. coli. III. The role of internucleotide phosphate groups of the matrix in its binding with the enzyme]. 266 77

The values of Kd and Gibbs energy (delta G degrees) have been measured for complexes of the template site of DNA polymerase I Klenow fragment with the homo-oligonucleotides d(pC)n, d(pT)n, and d(pA)n and hetero-oligonucleotides of various structures and lengths. These parameters were evaluated from the protective effect of the oligonucleotide on enzyme inactivation by the affinity reagents d(Tp)2C[Pt2+ (NH3)2OH](pT)7 and d[(Tp2)C(Pt2+(NH3)2OH)p]3T of the template site. The present results and previously reported data [(1985) Biorg. Khim. 13, 357-369] indicate that the nucleoside components of the template form complexes as a result of their hydrophobic interactions with the enzyme. Only one template internucleotide phosphate forms an Me2+-dependent electrostatic contact and a hydrogen bond with the enzyme. The 19-20-nucleotide fragments of the template appear to interact with the protein molecule.
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PMID:DNA polymerase I (Klenow fragment): role of the structure and length of a template in enzyme recognition. 272 84

Helenalin and bis (helenalinyl) malonate, sesquiterpene lactones, were shown to be cytotoxic against the growth of P-388 lymphocytic leukemia cells in culture. DNA and protein synthesis were reduced by these agents preferentially, with RNA synthesis being affected only marginally. This study focused on the identification of the enzyme target(s) responsible for the inhibition of DNA synthesis by the sesquiterpene lactones. Purine synthesis was strongly inhibited at the IMP dehydrogenase step. Suppression of IMP dehydrogenase activity and purine synthesis paralleled the DNA synthesis inhibition with respect to both dose dependence and time of incubation with drug. Deoxyribonucleoside triphosphate pools in the P-388 cells were significantly reduced by both drugs and the DNA polymerase alpha activity was only moderately inhibited by both drugs in cytoplasmic preparation. However, inhibition of a partially purified DNA polymerase alpha was of a much greater magnitude. Activity of the ribonucleotide reductase complex was reduced by more than 50% at 100 microM concentration of either drug. The drugs appeared to affect the hydrogen donor system of the reductase complex, since the activity of the ribonucleotide reductase enzyme itself was not affected but both thioredoxin and glutaredoxin were markedly inactivated by the sesquiterpene lactones. Thymidylate synthetase activity was not affected by the sesquiterpene lactones in P-388 cells. These data suggest that the inhibition of IMP dehydrogenase and the ribonucleotide reductase complex activities by helenalin and bis (helenalinyl) melonate was the primary reason for the observed inhibition of DNA synthesis, but that inhibition of DNA polymerase alpha may also play a role. The inhibition of the sensitive enzymes is likely to be related to drug alkylation of thiol active groups of the enzymes in a manner similar to the action of N-ethylmaleimide. The mode of action of helenalin and bis (helenalinyl) malonate does not appear to be similar to that of the parthenolide-type sesquiterpene lactones which contain an epoxide moiety.
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PMID:Inhibition of nucleic acid synthesis in P-388 lymphocytic leukemia cells in culture by sesquiterpene lactones. 289 92

The adenovirus-encoded 140-kDa DNA polymerase (Ad Pol) and the 59-kDa DNA binding protein (Ad DBP) are both required for the replication of viral DNA in vivo and in vitro. Previous studies demonstrated that, when poly(dT).oligo(dA) was used as a template-primer, both proteins were required for poly(dA) synthesis. In this report, the interaction between the Ad Pol and Ad DBP was further investigated using poly(dT).oligo(dA) as well as a linear duplex molecule containing 3' poly(dT) tails. DNA synthesis with the tailed template required Ad Pol, Ad DBP, and an oligo(dA) primer hydrogen bonded to the poly(dT) tails. Incorporation was stimulated 8-10-fold by ATP; however, no evidence of ATP hydrolysis to ADP was observed. Synthesis was initiated at either end of the tailed molecule and proceeded through the duplex region to the end of the molecule. This ability to translocate through duplex DNA and to synthesize long poly(dA) chains suggests that the Ad Pol.Ad DBP complex can act efficiently in the elongation reactions involved in the replication of Ad DNA (both type I and type II). During the replication reaction, substantial hydrolysis of deoxynucleoside triphosphates to the corresponding deoxynucleoside monophosphates occurred. This reaction required DNA synthesis and most likely reflects an idling reaction similar to that observed with other DNA polymerases containing 3'----5' exonuclease activity in which the polymerase first incorporates and then hydrolyzes a dNMP.
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PMID:The adenovirus DNA binding protein and adenovirus DNA polymerase interact to catalyze elongation of primed DNA templates. 294 38


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