Abstract
A variety of biochemical and structural studies indicate that two regions of the human immunodeficiency virus type 1 (HIV-1) polypurine tract (PPT)-containing RNA/DNA hybrid deviate from standard Watson-Crick geometry. However, it is unclear whether and how these regions cooperate to ensure PPT primer selection by reverse transcriptase-associated ribonuclease H and subsequent removal from nascent (+)-DNA. To address these issues, we synthesized oligonucleotides containing abasic lesions in either the PPT (+)-RNA primer or (-)-DNA template to locally remove nucleobases, although retaining the sugar-phosphate backbone. KMnO(4) footprinting indicates such lesions locally alter duplex structure, whereas thermal melting studies show significantly reduced stability when lesions are positioned around the scissile bond. Substituting the (-)-DNA template between positions -15 and -13 altered cleavage specificity, whereas equivalent substitutions of the (+)-RNA had almost no effect. The unpaired base of the DNA template observed crystallographically (-11C) could also be removed without significant loss of cleavage specificity. With respect to the scissile -1/+1 phosphodiester bond, template nucleobases could be removed without loss of cleavage specificity, whereas equivalent lesions in the RNA primer were inhibitory. Our data suggest an interaction between the p66 thumb subdomain of HIV-1 reverse transcriptase, and the DNA template in the "unzipped" portion of the RNA/DNA hybrid could aid in positioning the ribonuclease H catalytic center at the PPT/U3 junction and also provides insights into nucleic acid geometry around the scissile bond required for hydrolysis.
Highlights
From the Reverse Transcriptase Biochemistry Section, Resistance Mechanisms Laboratory, HIV Drug Resistance Program, NCI, National Institutes of Health, Frederick, Maryland 21702
Our data suggest an interaction between the p66 thumb subdomain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, and the DNA template in the “unzipped” portion of the RNA/DNA hybrid could aid in positioning the ribonuclease H catalytic center at the PPT/U3 junction and provides insights into nucleic acid geometry around the scissile bond required for hydrolysis
The second region selected for analysis was the Ϫ1/ϩ1 scissile bond corresponding to the PPT/U3 junction, where lesions were introduced on either side of the junction and downstream at position ϩ2
Summary
Vol 280, No 20, Issue of May 20, pp. 20154 –20162, 2005 Printed in U.S.A. Investigating HIV-1 Polypurine Tract Geometry via Targeted Insertion of Abasic Lesions in the (؊)-DNA Template and (؉)-RNA Primer*. We and others have noted that template base ϩ1 of the HIV-1 and murine leukemia virus PPT is surprisingly tolerant to substitution, accepting substitution with non-hydrogenbonding pyrimidine isosteres and base mismatches with minimal effect on the accuracy and overall rate of hydrolysis [2, 9, 13] The latter observations might indicate that, as the RNA/ DNA hybrid enters the RNase H catalytic center, the DNA strand is displaced, allowing “docking” of the scissile phosphodiester bond in the active site, a model consistent with the role proposed for the p66 RNase H primer grip [8]. With respect to structural requirements at the PPT/U3 junction, we show here that, eliminating primer bases 5Ј and 3Ј of the scissile bond destroys cleavage, removing their complement on the (Ϫ)-DNA template is only partially inhibitory, indicating that, consistent with studies on human RNase H1 [26], a DNA base at the scissile bond is not essential for hydrolysis
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