title: Apparent defects in processive DNA synthesis, strand transfer, and primer elongation of Met-184 mutants of HIV-1 reverse transcriptase derive solely from a dNTP utilization defect.
abstract: The 2',3'-dideoxy-3'-thiacytidine drug-resistant M184I HIV-1 reverse transcriptase (RT) has been shown to synthesize DNA with decreased processivity compared with the wild-type RT. M184A displays an even more severe processivity defect. However, the basis of this decreased processivity has been unclear, and both primer-template binding and dNTP interaction defects have been proposed to account for it. In this study, we show that the altered properties of the M184I and M184A RT mutants that we have measured, including decreased processivity, a slower rate of primer extension, and increased strand transfer activity, can all be explained by a defect in dNTP utilization. These alterations are observed only at low dNTP concentration and vanish as the dNTP concentration is raised. The mutant RTs exhibit a normal dissociation rate from a DNA primer-RNA template while paused during synthesis. Slower than normal synthesis at physiological dNTP concentration, coupled with normal dissociation from the primer-template, results in the lowered processivity. The mutant RTs exhibit normal DNA 3'-end-directed and RNA 5'-end-directed ribonuclease H activity. The reduced rate of DNA synthesis causes an increase in the ratio of ribonuclease H to polymerase activity thereby promoting increased strand transfer. These latter results are consistent with an observed higher rate of recombination by HIV-1 strains with Met-184 mutations.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY 14642, USA.
authors.forename: Lu
authors.initials: L
authors.lastname: Gao
authors.forename: Mark Nils
authors.initials: MN
authors.lastname: Hanson
authors.forename: Mini
authors.initials: M
authors.lastname: Balakrishnan
authors.forename: Paul L
authors.initials: PL
authors.lastname: Boyer
authors.forename: Bernard P
authors.initials: BP
authors.lastname: Roques
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Hughes
authors.forename: Baek
authors.initials: B
authors.lastname: Kim
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bambara
mesh_terms: D019943:Amino Acid Substitution; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D003854:Deoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism; D024882:Drug Resistance, Viral / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D019259:Lamivudine / Q000737:chemistry / Q000494:pharmacology; D020125:Mutation, Missense; D011995:Recombination, Genetic / Q000187:drug effects / Q000235:genetics; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D054309:Ribonuclease H, Human Immunodeficiency Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D003854:Deoxyribonucleotides; D018894:Reverse Transcriptase Inhibitors; D019259:Lamivudine; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D054309:Ribonuclease H, Human Immunodeficiency Virus