Incorporation of the guanosine triphosphate analogs 8-oxo-dGTP and 8-NH2-dGTP by reverse transcriptases and mammalian DNA polymerases.
J Biol Chem
; 272(9): 5892-8, 1997 Feb 28.
Article
em En
| MEDLINE
| ID: mdl-9038207
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 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.
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Base de dados:
MEDLINE
Assunto principal:
DNA Polimerase Dirigida por RNA
/
Nucleotídeos de Desoxiguanina
/
Mutagênicos
Idioma:
En
Ano de publicação:
1997
Tipo de documento:
Article