Utilization of 2',3'-dideoxyguanosine 5'-triphosphate as an inhibitor and substrate for DNA polymerase alpha.
The activity of DNA polymerase alpha was strongly inhibited by low concentrations (less than 1 microM) of 2',3'-dideoxyguanosine 5'-triphosphate (ddGTP) in the presence of Mn2+, although the incorporation of [3H]ddGTP into the product DNA was not detectable under the same reaction conditions. ddGTP competitively inhibited the incorporation of [3H]dGTP into the DNA. The Kis of DNA polymerase alpha for ddGTP were as low as 0.035 microM with activated DNA and 0.044 microM with (dC)n.(dG)12-18 as respective template.primers. By increasing [3H]ddGTP concentration (greater than 1 microM), however, it became an efficient substrate for DNA polymerase alpha. Under the assay conditions with activated DNA as the template.primer and Mg2+ as the divalent cation, the maximum incorporation rate of [3H]ddGTP reached 14.5% when compared to that of the corresponding normal substrate [3H]dGTP. These results indicate that the observed inhibition of DNA polymerase alpha activity by low concentrations of ddGTP in the presence of Mn2+ cannot be explained by the incorporation of ddGTP followed by chain termination of the growing DNA, and the results also support the previously presented hypothesis that the inhibition is caused by competition between the dideoxynucleotide inhibitor and its corresponding deoxynucleotide substrate at the same enzyme binding site. Thus ddGTP acts as an inhibitor or a substrate for DNA polymerase alpha depending on its concentration and the species of divalent cation. Some of the kinetic properties of DNA polymerase alpha connected with ddGTP are also described.
Ono
K
K
Laboratory of Viral Oncology, Aichi Cancer Center Research Institute, Nagoya, Japan.
Nakane
H
H
D001667:Binding, Competitive / Q000187:drug effects; D004257:DNA Polymerase II / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000493:pharmacokinetics; D004305:Dose-Response Relationship, Drug; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D013379:Substrate Specificity
D008345:Manganese; D004257:DNA Polymerase II; D008274:Magnesium