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J. Biol. Chem., Vol. 279, Issue 35, 37095-37102, August 27, 2004

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Two Modes of HIV-1 Polypurine Tract Cleavage Are Affected by Introducing Locked Nucleic Acid Analogs into the (-) DNA Template^*

Chandravanu Dash, Hye-Young Yi-Brunozzi, and Stuart F. J. Le Grice

From the Resistance Mechanisms Laboratory, HIV Drug Resistance Program, NCI-Frederick, National Institutes of Health, Frederick, Maryland 21702

Unusual base-pairing in a co-crystal of reverse transcriptase (RT) and a human immunodeficiency virus type 1 (HIV-1) polypurine tract (PPT)-containing RNA/DNA hybrid suggests local nucleic acid flexibility mediates selection of the plus-strand primer. Structural elements of HIV-1 RT potentially participating in recognition of this duplex include the thumb subdomain and the ribonuclease H (RNase H) primer grip, the latter comprising elements of the connection subdomain and RNase H domain. To investigate how stabilizing HIV-1 PPT structure influences its recognition, we modified the (-) DNA template by inserting overlapping locked nucleic acid (LNA) doublets and triplets. Modified RNA/DNA hybrids were evaluated for cleavage at the PPT/U3 junction. Altered specificity was observed when the homopolymeric dA·rU tract immediately 5' of the PPT was modified, whereas PPT/U3 cleavage was lost after substitutions in the adjacent dT·rA tract. In contrast, the "unzipped" portion of the PPT was moderately insensitive to LNA insertions. Although a portion of the dC·rG and neighboring dT·rA tract were minimally affected by LNA insertion, RNase H activity was highly sensitive to altering the junction between these structural elements. Using 3'-end-labeled PPT RNA primers, we also identified novel cleavage sites ahead (+5/+6) of the PPT/U3 junction. Differential cleavage at the PPT/U3 junction and U3 + 5/+6 site in response to LNA-induced template modification suggests two binding modes for HIV-1 RT, both of which may be controlled by the interaction of its thumb subdomain (potentially via the minor groove binding track) at either site of the unzipped region.

Received for publication, March 24, 2004 , and in revised form, June 23, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Reverse Transcriptase Biochemistry Section, HIV Drug Resistance Program, NCI-Frederick, MD 21702. Tel.: 301-846-5256; Fax: 301-846-6013; E-mail: slegrice{at}ncifcrf.gov.

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