Incorporation of the guanosine triphosphate analogs 8-oxo-dGTP and 8- NH2-dGTP by reverse transcriptases and mammalian DNA polymerases

Abstract

We have measured the efficiencies of utilization of 8-oxo-dGTP and 8- NH2-dGTP by human immunodeficiency virus type 1 and murine leukemia virus reverse transcriptases and compared them to those of DNA polymerases α and β. Initially, we carried out primer extension reactions in the presence of dGTP or a dGTP analog and the remaining three dNTPs using synthetic DNA and RNA templates. These assays revealed that, in general, 8-NH2-dGTP is incorporated and extended more efficiently than 8-oxo-dGTP by all enzymes tested. Second, we determined rate constants for the incorporation of each analog opposite a template cytidine residue using steady state single nucleotide extension kinetics. Our results demonstrated the following. 1) Both reverse transcriptases incorporate the nucleotide analogs; discrimination against their incorporation is a function primarily of K(m) or V(max) depending on the analog and the enzyme. 2) Discrimination against the analogs is more stringent with the DNA template than with a homologous RNA template. 3) Polymerase α exhibits a mixed kinetic phenotype, with a large discrimination against 8-oxo-dGTP but a comparatively higher preference for 8-NH2-dGTP. 4) Polymerase β incorporates both analogs efficiently; there is no discrimination with respect to K(m) and a significantly lower discrimination with respect to V(max) when compared with the other polymerases.