The sequential mechanism of HIV reverse transcriptase RNase H.
Synthesis of the minus strand of viral DNA by human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase is accompanied by RNase H degradation of the viral RNA genome. RNA fragments remain after synthesis and are degraded by the polymerase-independent mode of RNase H cleavage. Recently, we showed that this mode of cleavage occurs by a specific ordered mechanism in which primary cuts are first, secondary and 5-nucleotide cuts are next, and second primary cuts occur last (Wisniewski, M., Balakrishnan, M., Palaniappan, C., Fay, P., J., and Bambara, R., A. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 11978-11983). Ultimately the RNAs are cleaved into small fragments that can dissociate from the DNA template. Because the cleavage mechanism is an ordered series of events, we determined in this study whether any earlier cut is required for a later cut. By precisely inhibiting cleavage at each site, we examined the dependence of later cuts on cleavage at that site. We found that each cut is independent of the other cuts, demonstrating that the order of this stepwise mechanism is based on the rates of each cut. A mechanism for unlinked ordered cleavage consistent with these results is presented.
Wisniewski
M
M
Department of Biochemistry and Biophysics and the Cancer Center, University of Rochester, Rochester, New York 14642, USA.
Balakrishnan
M
M
Palaniappan
C
C
Fay
P J
PJ
Bambara
R A
RA
D001483:Base Sequence; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D008969:Molecular Sequence Data; D012313:RNA / Q000737:chemistry; D016914:Ribonuclease H / Q000378:metabolism*
D012313:RNA; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H