Two DNA polymerases of Escherichia coli display distinct misinsertion specificities for 2-hydroxy-dATP during DNA synthesis.
The insertion specificities of an oxidized dATP analogue, 2-hydroxydeoxyadenosine 5'-triphosphate (2-OH-dATP), were determined using the alpha (catalytic) subunit of Escherichia coli DNA polymerase III and the exonuclease-deficient Klenow fragment of DNA polymerase I. In contrast to our previous observation that mammalian DNA polymerase alpha incorporated the oxidized nucleotide opposite T and C, these two E. coli DNA polymerases incorporated 2-OH-dATP opposite T and G on the DNA template. Steady-state kinetic studies indicated that the alpha subunit incorporated 2-OH-dATP 10 times more frequently opposite T than opposite G. On the other hand, the incorporation of 2-OH-dATP opposite T by the exonuclease-deficient Klenow fragment was 2 orders of magnitude more efficient than that opposite G. These results indicate that the misinsertion specificity of 2-OH-dATP differs between replicative and repair-type DNA polymerases, and provide a biochemical basis for the mutations induced by 2-OH-dATP in E. coli.
Department of Environmental Oncology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Yahatanishi-ku, Kitakyushu, Japan.
H
H
Kamiya
H
H
Maki
H
H
Kasai
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D002384:Catalysis; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D004258:DNA Polymerase III / Q000378:metabolism*; D004261:DNA Replication; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000172:deficiency / Q000235:genetics; D007700:Kinetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
C095141:2-hydroxydeoxyadenosine triphosphate; D017382:Reactive Oxygen Species; D000255:Adenosine Triphosphate; D004256:DNA Polymerase I; D004258:DNA Polymerase III; D005090:Exodeoxyribonucleases