Revisiting Plus-Strand DNA Synthesis in Retroviruses and Long Terminal Repeat Retrotransposons: Dynamics of Enzyme: Substrate Interactions

Author:

Fabris, D.

Marino, J. P.

Le Grice, S. F. J.
Author Address

[Le Grice, Stuart F. J.] NCI, HIV Drug Resistance Program, NIH, Frederick, MD 21702 USA. [Fabris, Daniele] Univ Maryland Baltimore Cty, Dept Chem & Biochem, Catonsville, MD 21228 USA. [Marino, John P.] Univ Maryland, Inst Biotechnol, Ctr Adv Res Biotechnol, Rockville, MD 20850 USA. [Marino, John P.] NIST, Rockville, MD 20850 USA.;Le Grice, SFJ, NCI, HIV Drug Resistance Program, NIH, Frederick, MD 21702 USA.;legrices@mail.nih.gov

Abstract:

Although polypurine tract (PPT)-primed initiation of plus-strand DNA synthesis in retroviruses and LTR-containing retrotransposons can be accurately duplicated, the molecular details underlying this concerted series of events remain largely unknown. Importantly, the PPT 3' terminus must be accommodated by ribonuclease H (RNase H) and DNA polymerase catalytic centers situated at either terminus of the cognate reverse transcriptase (RT), and in the case of the HIV-1 enzyme, similar to 70 angstrom apart. Communication between RT and the RNA/DNA hybrid therefore appears necessary to promote these events. The crystal structure of the HIV-1 RT/PPT complex, while informative, positions the RNase H active site several bases pairs from the PPT/U3 junction, and thus provides limited information on cleavage specificity. To fill the gap between biochemical and crystallographic approaches, we review a multidisciplinary approach combining chemical probing, mass spectrometry, NMR spectroscopy and single molecule spectroscopy. Our studies also indicate that nonnucleoside RT inhibitors affect enzyme orientation, suggesting initiation of plus-strand DNA synthesis as a potential therapeutic target.

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