Uniquely altered DNA replication fidelity conferred by an amino acid change in the nucleotide binding pocket of human immunodeficiency virus type 1 reverse transcriptase.
Arginine 72 in human immunodeficiency virus type 1 reverse transcriptase (RT), a highly conserved residue among retroviral polymerases and telomerases, forms part of the binding pocket for the nascent base pair. We show here that replacement of Arg(72) by alanine strongly alters fidelity in a highly unusual manner. R72A reverse transcriptase is a frameshift and base substitution antimutator polymerase whose increased fidelity results both from increased nucleotide selectivity and from a decreased ability to extend mismatched primer termini. Thus, Arg(72)-substrate interactions in wild-type human immunodeficiency virus type 1 RT can stabilize incorrect nucleotides allowing misinsertion and promoting extension of mismatched and perhaps misaligned template-primers. In contrast to the higher fidelity at most sites, R72A RT is highly error-prone for misincorporations opposite template T in the sequence context: 5'-CTGG. Surprisingly, this results mostly from a 1200-fold increase in the apparent K(m) for correct dAMP incorporation. Thus, Arg(72) interactions with substrate are critical for the stability of the correct T.dAMP base pair when the 5'-CTGG sequence is present in the binding pocket for the nascent base pair. Collectively, the data show that a mutant polymerase may yield higher than normal average replication fidelity, yet paradoxically place specific sequences at very high risk of mutation.
Lewis
D A
DA
Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Bebenek
K
K
Beard
W A
WA
Wilson
S H
SH
Kunkel
T A
TA
D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D004261:DNA Replication; D003854:Deoxyribonucleotides / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D013379:Substrate Specificity; D013698:Templates, Genetic
D017931:DNA Primers; D003854:Deoxyribonucleotides; D054303:HIV Reverse Transcriptase