The base substitution fidelity of HIV-1 reverse transcriptase on DNA and RNA templates probed with 8-oxo-deoxyguanosine triphosphate.
We have used 8-O-dGTP, a mutagenic nucleotide generated by oxidative metabolism, to probe the misincorporation potential of HIV-1 reverse transcriptase (RT) during DNA synthesis templated by the same nucleotide sequence as either RNA or DNA. With either template, 8-O-dGMP was misincorporated opposite template A, yielding characteristic A-->C transversions. The error rate with DNA was similar to that with RNA, suggesting that base misincorporation by the RT during first-strand and second-strand replication may contribute equally to the HIV-1 base substitution mutation rate. The rate of 8-O-dGMP misincorporation differed by more than 10-fold among the 20 adenines in the M13mp2 template where A-->C transversions can be detected. The transversion distribution was similar with the two templates, indicating that the effects of flanking nucleotides on misincorporation rates were similar. This is consistent with structural and biochemical data suggesting that HIV-1 RT binds RNA x DNA and DNA x DNA template-primers in the same orientation. The similarities in error rates and distribution further indicate that, despite differences in the structures of free RNA x DNA and DNA x DNA duplexes (e.g., minor groove dimensions), the polymerase active site that assembles upon substrate binding establishes a similar degree of nucleotide selectivity with both types of template-primers. Comparison of the RT error distribution to that observed with two Pol I family DNA polymerases and a Pol alpha family polymerase revealed common hot and cold spots for misincorporation. This suggests that the local nucleotide sequence influences the nucleotide selectivity of four polymerases in a similar manner, despite their differences in structure, biochemical properties, and functions.
Bebenek
K
K
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709, USA.
Boyer
J C
JC
Kunkel
T A
TA
D020137:Base Pair Mismatch; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000096:biosynthesis* / Q000235:genetics; D003848:Deoxyguanine Nucleotides; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes; D012313:RNA / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D013698:Templates, Genetic
D003848:Deoxyguanine Nucleotides; D009692:Nucleic Acid Heteroduplexes; D011994:Recombinant Proteins; C078206:8-oxodeoxyguanosine triphosphate; D012313:RNA; D004247:DNA; D054303:HIV Reverse Transcriptase