Misincorporation and mispaired primer extension by human immunodeficiency virus reverse transcriptase.
Pre-steady-state methods were used to study the fidelity of human immunodeficiency virus reverse transcriptase. Fidelity of DNA-directed DNA synthesis can be attributed to a 1-2 order of magnitude reduction in affinity for noncomplementary dNTPs, and a 1-4 order of magnitude reduction in the rate of the conformational change that limits the rate of nucleotide addition. Affinities of reverse transcriptase for paired or mispaired primer termini are similar. Discrimination against a mispaired primer is due to reduction in affinity for the next dNTP and reduction in rate of extension. Extension of mispaired termini proceeds 20-700-fold faster than the rate of dissociation of reverse transcriptase from the primer-template and is 2-3 orders of magnitude more frequent than nucleotide misincorporation. The rate-limiting step for extension of a mispaired terminus occurs at the conformational change or chemical step, depending on the nature of the mispair. Presence of a mismatch at the 3' penultimate position reduces pyrophosphorolysis of the primer by a factor of 10(3), indicating that mispairs 5' to the site of chemistry can also affect catalysis.
Zinnen
S
S
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Hsieh
J C
JC
Modrich
P
P
D001482:Base Composition; D001483:Base Sequence; D017931:DNA Primers; D004261:DNA Replication; D006678:HIV / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D013698:Templates, Genetic
D017931:DNA Primers; D009692:Nucleic Acid Heteroduplexes; D009711:Nucleotides; D012194:RNA-Directed DNA Polymerase