title: Rapid kinetic analysis of a point mutant of HIV-1 reverse transcriptase lacking ribonuclease H activity.
abstract: The comparative kinetics of RNA-dependent DNA polymerization catalyzed by wild-type HIV-1 reverse transcriptase and a point mutant specifically lacking RNase H activity were analyzed using a heteropolymeric substrate consisting of a 19-mer primer hybridized to a 345-nucleotide RNA template. The rapid-quench product distributions generated under single-turnover conditions, in which primer extension by the two enzymes was restricted to the incorporation of 5 nucleotides (N+5), were significantly different. Whereas the wild-type enzyme catalyzed synthesis of the N+5 product over the time course of the reaction (20 ms-10 s) with a relatively low degree of processivity, the extent of accumulation of the intermediate N+2 and N+3 products was grossly exaggerated in the parallel mutant-catalyzed time course. The observation of concomitant polymerase-dependent hydrolysis during the course of synthesis catalyzed by the wild-type enzyme suggested that the inability of the RNase H- mutant to hydrolyze the RNA template created blocks to further synthesis by reducing the rates of DNA polymerization at these intermediate positions, and hence impaired the ability of this mutant to complete cDNA synthesis.
authors.affiliation: South African Institute for Medical Research, Department of Haematology, University of the Witwatersrand Medical School, Johannesburg.
authors.forename: L R
authors.initials: LR
authors.lastname: Dudding
authors.forename: V
authors.initials: V
authors.lastname: Mizrahi
mesh_terms: D001483:Base Sequence; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D017354:Point Mutation; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism*; D013698:Templates, Genetic
chemical_list: D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H