Requirements for DNA unpairing during displacement synthesis by HIV-1 reverse transcriptase.
DNA displacement synthesis by reverse transcriptase during retroviral replication is required for the production of the linear precursor to integration. The sensitivity of unpaired thymines to KMnO(4) oxidation was used to probe for the extent of DNA melting by human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase in front of the primer terminus in model oligonucleotide-based displacement constructs. Unpairing of the two base pairs downstream of the primer (+1 and +2 positions) requires the presence of the next correct dNTP, indicating that DNA melting only occurs after the formation of the ternary complex with the enzyme tightly clamped around the DNA. The amount or extent of DNA melting is not significantly affected by the length of the already-displaced strand or the base composition of the DNA beyond the +2 position. The F61W mutant form of HIV-1 reverse transcriptase, which is partially impaired for displacement synthesis, exhibits a reduction in the amount of melting at the +1 and +2 positions. These results demonstrate the importance of the observed melting to displacement synthesis and suggest that the unpairing reaction is mediated by an intimate association between the fingers region of the enzyme and the DNA in the closed clamp conformation of the protein.
Winshell
Jamie
J
Department of Microbiology, School of Medicine, University of Washington, Seattle, WA 98195-7242, USA.
Paulson
Benjamin A
BA
Buelow
Ben D
BD
Champoux
James J
JJ
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D011196:Potassium Permanganate / Q000737:chemistry*; D013941:Thymine / Q000737:chemistry; D013997:Time Factors
D017931:DNA Primers; D004279:DNA, Viral; D009841:Oligonucleotides; D011196:Potassium Permanganate; D004247:DNA; D054303:HIV Reverse Transcriptase; D013941:Thymine