Human immunodeficiency virus reverse transcriptase displays a partially processive 3' to 5' endonuclease activity.
We have examined the ribonuclease H (RNase H) activity of human immunodeficiency virus reverse transcriptase (HIV-RT) using a natural sequence 83-nucleotide-long RNA template to which was hybridized a DNA oligomer. This substrate configuration allowed for the simultaneous electrophoretic resolution of 5'-, 3'-, and internally derived RNase H cleavage products. Assays performed in the presence of excess challenger RNA to sequester the RT permitted the analysis of products resulting from a single round of binding of RT to substrate. Substrate cleavage was highly sensitive to ionic strength, showing greatest activity at low KCl concentrations. The increase in cleavage correlated with an increase in the half-life of the enzyme on the RNA-DNA hybrid from approximately 31 s to 6.2 min at 80 and 5 mM KCl, respectively. Internally derived cleavage products generated in challenged reactions were primarily 2-9 nucleotides in length. These lengths indicate that the products were generated by an endo- rather than an exonuclease activity. The directionality and processivity of the endonuclease were also determined by examination of cleavage products from challenged reactions. Although the lengths of 5'-derived products markedly decreased with time, no change in the size distribution of 3'-derived products was observed, indicating that cleavage proceeded processively in the 3' to 5' direction. The 5'-derived products were shortened more in reactions performed under conditions allowing multiple versus single enzyme-binding events, suggesting that the endonuclease action of a single enzyme is not processive enough to generate the maximum possible amount of cleavage on each substrate. Therefore, HIV-RT displays a partially processive 3' to 5' endonuclease activity.
DeStefano
J J
JJ
Department of Biochemistry, University of Rochester, New York 14642.
Buiser
R G
RG
Mallaber
L M
LM
Bambara
R A
RA
Fay
P J
PJ
D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases / Q000378:metabolism*; D006678:HIV / Q000201:enzymology*; D006207:Half-Life; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012313:RNA / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism*; D013379:Substrate Specificity; D014158:Transcription, Genetic
D012313:RNA; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D004720:Endonucleases; D016914:Ribonuclease H