Incorporation of the guanosine triphosphate analogs 8-oxo-dGTP and 8-NH2-dGTP by reverse transcriptases and mammalian DNA polymerases.
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 alpha and beta. 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 Km or Vmax 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 alpha exhibits a mixed kinetic phenotype, with a large discrimination against 8-oxo-dGTP but a comparatively higher preference for 8-NH2-dGTP. 4) Polymerase beta incorporates both analogs efficiently; there is no discrimination with respect to Km and a significantly lower discrimination with respect to Vmax when compared with the other polymerases.
Kamath-Loeb
A S
AS
Department of Pathology, University of Washington, Seattle, Washington 98195, USA.
Hizi
A
A
Kasai
H
H
Loeb
L A
LA
D004257:DNA Polymerase II / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism*; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009052:Leukemia Virus, Murine / Q000201:enzymology; D009153:Mutagens / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013698:Templates, Genetic
C105095:8-amino-2-deoxyguanosine triphosphate; D003848:Deoxyguanine Nucleotides; D009153:Mutagens; C078206:8-oxodeoxyguanosine triphosphate; C029603:deoxyguanosine triphosphate; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D004257:DNA Polymerase II