CHARACTERIZATION OF AN RNASE-H DEFICIENT MUTANT OF HUMAN IMMUNODEFICIENCY VIRUS-1 REVERSE-TRANSCRIPTASE HAVING AN ASPARTATE TO ASPARAGINE CHANGE AT POSITION-498

It has been proposed that Asp-443, Glu-478, and Asp-498 are important in RNase H mediated catalysis by human immunodeficiency virus-1 revers e 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., Us din, M.T., Harington, A. and Dudding, L.R. (1990) Nucleic Acids Res. 1 8, 5359-5363) or 478 (Schatz, O., Cromme, F.V., Gruninger-Leitch, F. a nd Le Grice, S.F.J. (1989) FEBS Lett. 257, 311-314) severely inhibit R Nase H activity but have only small effects on polymerase activity. We show here that a single mutation at position 498 of Asp to Asn (mutan t D498N) results in a stable enzyme with a 20-fold reduction in the ra tio of RNase H to polymerase activity. The mutant and wild type enzyme s 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(r A) was also the same. The results indicate that the mutation does not affect normally measured catalytic parameters of the polymerase functi on of the enzyme. D498N catalyzed strand transfer synthesis on homopol ymeric, but not heteropolymeric templates, indicating that RNase H act ivity is not required for the former activity, but is for the latter.