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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of single and combined heat treatments on the activity of DNA polymerase beta was studied in CHO cells. The activity of polymerase beta was determined by measuring the amount of [3H]TTP incorporated into activated calf thymus DNA in the presence of aphidicolin, a specific inhibitor of DNA polymerase alpha. Biphasic response curves were obtained for all temperatures tested (40-46 degrees C) showing the sensitivity to decrease during heating. A constant activation energy of Ea = 120 +/- 10 kcal/mole was found for the initial heat sensitivity, whereas the Arrhenius plot for the final sensitivity is characterized by an inflection point at 43 degrees C with Ea = 360 +/- 40 kcal/mole or Ea = 130 +/- 20 kcal/mole for temperatures below or above 43 degrees C, respectively. The observed decrease of the polymerase activity is not due to a decrease in the number of active enzyme molecules but to a change in its affinity, since the inhibition is reversible when increasing concentrations of TTP are applied. When acute or chronic thermo-tolerance was induced by a priming heat treatment at 43 degrees C for 45 min followed by a time interval at 37 degrees C for 16 h or by a preincubation at 40 degrees C for 16 h, respectively, the thermal sensitivity of polymerase beta was lowered by a factor of up to 5. By contrast, pretreatment at a higher temperature followed by a lower temperature (step-down heating) did not alter the sensitivity of polymerase beta to the second treatment. The results indicate that heat-induced cell death cannot be the consequence of the reduction of the polymerase beta activity, confirming earlier studies on this subject.
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PMID:Reduction of DNA-polymerase beta activity of CHO cells by single and combined heat treatments. 350 Jan 45

The affinity of different ligands (phosphate, nucleoside monophosphates, oligonucleotides) to the template binding site of DNA polymerase alpha from human placenta was estimated. To this goal, dependences of rate of the enzyme inactivation by the affinity reagent d(pT)2pC[Pt2+(NH3)2OH](pT)7 on the concentration of these ligands as competitive inhibitors were determined. Minimal ligands capable to bind with the template site of DNA polymerase alpha were shown to be triethylphosphate (Kd 600 microM) and phosphate (Kd 53 microM). Ligand affinity increases by the factor 1.71 per added monomer unit from phosphate to d(pT) and then for oligothymidylates d(Tp)nT (n 1 to 14). The partial ethylation of phosphodiester groups does not change the efficiency of the oligothymidylate binding with the enzyme. However, the complete ethylation of these groups lowers affinity of the oligothymidylates to the enzyme by 7-9 times. The decrease is comparable with the change of Pt2+-decathymidylate affinity to the enzyme caused by Mn2+-ions. The data obtained led to suggestion that an electrostatic contact (most likely, Me2+-dependent) of phosphodiester group with the enzyme takes place. The type of contact is confirmed by Gibbs' energy change 1.1-1.4 kcal/mole. Formation of a hydrogen bond with the oxygen atom of P = O group of the same phosphate is also assumed (delta G =--4.4 . . .--4.5 kcal/mole). The other internucleotide phosphates and all bases of oligonucleotides form neither hydrogen bonds nor electrostatic contacts with the template binding site. Gibbs' energy changes by 0.32 kcal/mole when the template is lengthened by one unit. We suppose that this value characterizes the energy gain in the transition of oligonucleotide template from aquous medium to the hydrophobic environement of the enzyme active site. Comparison of Km values of oligothymidylates and their partially or completely ethylated analogues as templates in the reaction of DNA polymerization catalysed by DNA polymerase alpha from human placenta and Klenow's fragment of E. coli DNA polymerase I suggests a similar mechanism of template recognition by both enzymes.
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PMID:[Eukaryotic and prokaryotic DNA-polymerase. II. The role of internucleotide phosphate groups of a template in its binding with the enzyme]. 355 64

Modification of human placenta DNA polymerase alpha by (pT)2pC[Pt2 + (NH3)2OH].(pT)7 was investigated. The linear time dependence of the enzyme activity logarithm suggested a pseudo-first order for modification. Kd value of enzyme-affinity reagent complex (0.5 microM) was estimated. The enzyme inactivation by the affinity reagent and protection from inactivation in the presence of oligonucleotides of varying length were used for determining Kd values of the enzyme-ligand complexes. Oligonucleotide d(pT)2pC(pT)7 (Kd 0.15 microM), d(Tp)9T (Kd 0.15 microM) and [d(Tp)9]ddT (Kd 0.15 microM) protected the enzyme from inactivation with equal efficiency. The protective action of oligothymidylates d(Tp)nT (where n changes from 3 to 14) strongly depended on the chain length, the Kd values diminishing from 5.3 to 0.0091 microM in the geometrical progression. The addition of one link to the oligothymidylate chain resulted in 1.71-fold increase in the oligonucleotide affinity for the enzyme specific site. Such a change corresponds to Gibbs energy change of about 0.32 kcal/mole. It is supposed that the monomer units of pentadecathymidylate (at least beginning with the third one) in d(Tp)14T-enzyme complex form neither hydrogen bonds nor electrostatic linkages with the enzyme. Kd values of oligonucleotides as templates are shown to reflect quite well the true affinity of template for the enzyme. This affinity increases in the presence of a primer. However, the ratio of the affinity for different oligonucleotides does not change in the presence or absence of a complementary primer.
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PMID:[DNA-polymerase alpha from human placenta. Effectiveness of interaction between oligothymidylates of different lengths and the template-binding site]. 396 8

The interactions of simian virus 40 (SV40) large T antigen with DNA carrying the viral origin of DNA replication, as well as its interactions with cellular replication proteins, have been investigated by using fluorescent ATP analogues as specific probes. The enhanced fluorescence of 3'(2')-O-(2,4, 6-trinitrophenyl)adenosine diphosphate (TNP-ADP) induced by T antigen binding to the nucleotide was decreased upon binding of T antigen to origin DNA. Similarly, the enhanced fluorescence induced by T antigen binding to TNP-ADP or TNP-ATP was decreased upon binding to human DNA polymerase alpha-primase (pol alpha), but not to replication protein A (RPA). Fluorescence titrations revealed noncompetitive inhibition of TNP-ADP binding by origin DNA, and noncompetitive inhibition of TNP-ADP and TNP-ATP binding by pol alpha, suggesting that T antigen complexed with either origin DNA or pol alpha was not able to bind the TNP nucleotide. From these titrations, we have measured a binding stoichiometry of 11.5 +/- 0.8 T antigen monomers per viral origin DNA, in agreement with the double hexamer assembly of T antigen on the origin as reported earlier. The stoichiometry of pol alpha binding to T antigen was measured to be 5.5 +/- 0.6 mol of T antigen per mole of pol alpha. While monomeric T antigen-nucleotide complex was a preferred ligand over free T antigen in the double hexamer assembly reaction, preformed T antigen hexamers were incapable of forming double hexamers on the DNA. The results support a model in which double hexamer assembly on the viral origin occurs by successive binding of 12 free T antigen or monomeric T-nucleotide complexes to the DNA. In contrast with this stepwise assembly of T antigen monomers on DNA, hexameric T antigen was able to bind directly to pol alpha with concomitant release of the bound TNP nucleotide. The possible implications of these results for the mechanism of initiation of SV40 DNA replication are discussed.
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PMID:Stoichiometry and mechanism of assembly of SV40 T antigen complexes with the viral origin of DNA replication and DNA polymerase alpha-primase. 979 95

The phylogenetic position of turtles is a currently controversial issue. Recent molecular studies rejected a traditional view that turtles are basal living reptiles (Hedges, S. B., and L. L. Poling. 1999. A molecular phylogeny. Science 83:998-1001; Kumazawa, Y., and M. Nishida. 1999. Complete mitochondrial DNA sequences of the green turtle and blue-tailed mole skink, statistical evidence for archosaurian affinity of turtles. Mol. Biol. Evol. 16:784-792). Instead, these studies grouped turtles with birds and crocodiles. The relationship among turtles, birds, and crocodiles remained unclear to date. To resolve this issue, we have cloned and sequenced two nuclear genes encoding the catalytic subunit of DNA polymerase alpha and glycinamide ribonucleotide synthetase-aminoimidazole ribonucleotide synthetase-glycinamide ribonucleotide formyltransferase from amniotes and an amphibian. The amino acid sequences of these proteins were subjected to a phylogenetic analysis based on the maximum likelihood method. The resulting tree showed that turtles are the sister group to a monophyletic cluster of archosaurs (birds and crocodiles). All other possible tree topologies were significantly rejected.
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PMID:Sister group relationship of turtles to the bird-crocodilian clade revealed by nuclear DNA-coded proteins. 1562 85