Protein-nucleic acid interaction in reactions catalyzed with DNA polymerases.
The affinities of oligothymidylates and of some analogs for the template site, of a set of oligodeoxyribo- and oligoribonucleotides for the primer site, and of dNTPs and some analogs for the substrate sites of DNA polymerase I Klenow fragment and of human placenta DNA polymerase alpha were measured using them either as competitors of affinity modification or as substrates. The data obtained enable us to hypothesize that the Me2+-dependent electrostatic contact and hydrogen bond of a single internucleotide phosphate and the hydrophobic interactions of the other nucleotide units determine the formation of oligonucleotide-template site complexes. Interaction of the primer's 3'-terminal hydroxy group and of the negatively charged adjacent phosphate with the enzyme, and Watson-Crick base pairing with the template are of crucial importance for the formation of the ternary enzyme-template-primer complex. dNTP and dNMP imidazolides inactivate enzymes via an affinity modification mechanism only in the presence of the template-primer complex. dNTP affinities exceed those of dNDPs and dNMPs, the enhancement being most significant for the substrate that is complementary to the template, thus suggesting the participation of the gamma-phosphate of dNTP in the substrate selection step.
D001667:Binding, Competitive; D050260:Carbohydrate Metabolism; D004256:DNA Polymerase I / Q000378:metabolism; D004257:DNA Polymerase II / Q000037:antagonists & inhibitors / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004563:Electrochemistry; D006801:Humans; D006860:Hydrogen Bonding; D008345:Manganese / Q000494:pharmacology; D009696:Nucleic Acids / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D010920:Placenta / Q000201:enzymology; D011506:Proteins / Q000378:metabolism*; D013698:Templates, Genetic; D013816:Thermodynamics
D009696:Nucleic Acids; D009711:Nucleotides; D009841:Oligonucleotides; D010710:Phosphates; D011506:Proteins; D008345:Manganese; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase