title: Characterization of an RNase H deficient mutant of human immunodeficiency virus-1 reverse transcriptase having an aspartate to asparagine change at position 498.
abstract: It has been proposed that Asp-443, Glu-478, and Asp-498 are important in RNase H mediated catalysis by human immunodeficiency virus-1 reverse transcriptase (Davies J.F., Hostomska, Z. Hostomsky, Z., Jordan, S.R. and Matthews, D.A. (1991) Science 251, 88-95; Mizrahi, V., Usdin, M.T., Harington, A. and Dudding, L.R. (1990) Nucleic Acids Res. 18, 5359-5363). Single point mutations at either position 443 (Mizrahi, V., Usdin, M.T., Harington, A. and Dudding, L.R. (1990) Nucleic Acids Res. 18, 5359-5363) or 478 (Schatz, O., Cromme, F.V., Grüninger-Leitch, F. and Le Grice, S.F.J. (1989) FEBS Lett. 257, 311-314) severely inhibit RNase H activity but have only small effects on polymerase activity. We show here that a single mutation at position 498 of Asp to Asn (mutant D498N) results in a stable enzyme with a 20-fold reduction in the ratio of RNase H to polymerase activity. The mutant and wild type enzymes were equally processive, paused in the same locations, and extended primers at the same rate during DNA synthesis on a heteropolymeric RNA template. The rate of elongation on the homopolymeric template poly(rA) was also the same. The results indicate that the mutation does not affect normally measured catalytic parameters of the polymerase function of the enzyme. D498N catalyzed strand transfer synthesis on homopolymeric, but not heteropolymeric templates, indicating that RNase H activity is not required for the former activity, but is for the latter.
authors.affiliation: Department of Biochemistry, University of Rochester, NY 14642.
authors.forename: J J
authors.initials: JJ
authors.lastname: DeStefano
authors.forename: W
authors.initials: W
authors.lastname: Wu
authors.forename: J
authors.initials: J
authors.lastname: Seehra
authors.forename: J
authors.initials: J
authors.lastname: McCoy
authors.forename: D
authors.initials: D
authors.lastname: Laston
authors.forename: E
authors.initials: E
authors.lastname: Albone
authors.forename: P J
authors.initials: PJ
authors.lastname: Fay
authors.forename: R A
authors.initials: RA
authors.lastname: Bambara
mesh_terms: D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001483:Base Sequence; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D008969:Molecular Sequence Data; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism*; D013379:Substrate Specificity; D013698:Templates, Genetic
chemical_list: D001224:Aspartic Acid; D001216:Asparagine; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H