Site-directed mutagenesis of the conserved Asp-443 and Asp-498 carboxy-terminal residues of HIV-1 reverse transcriptase.
Substitution of the conserved Asp-443 residue of HIV-1 reverse transcriptase by asparagine specifically suppressed the ribonuclease H activity of the enzyme without affecting the reverse transcriptase activity, suggesting involvement of this ionizable residue at the ribonuclease H active site. An analogous asparagine substitution of the Asp-498 residue yielded an unstable enzyme that was difficult to enzymatically characterize. However, the instability caused by the Asn-498 mutation was relieved by the introduction of a second distal Asn-443 substitution, yielding an enzyme with wild type reverse transcriptase activity, but lacking ribonuclease H activity.
Molecular Biology Laboratory, School of Pathology, South African Institute for Medical Research, Johannesburg.
V
V
Mizrahi
M T
MT
Usdin
A
A
Harington
L R
LR
Dudding
D000595:Amino Acid Sequence; D001216:Asparagine; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012194:RNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D016914:Ribonuclease H
D001216:Asparagine; D012194:RNA-Directed DNA Polymerase; D004722:Endoribonucleases; D016914:Ribonuclease H