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Mechanism of polypurine tract primer generation by HIV-1 reverse transcriptase.

Mechanism of polypurine tract primer generation by HIV-1 reverse transcriptase.
Author Information (click to view)

Figiel M, Krepl M, Park S, Poznański J, Skowronek K, Gołąb A, Ha T, Šponer J, Nowotny M,


Figiel M, Krepl M, Park S, Poznański J, Skowronek K, Gołąb A, Ha T, Šponer J, Nowotny M, (click to view)

Figiel M, Krepl M, Park S, Poznański J, Skowronek K, Gołąb A, Ha T, Šponer J, Nowotny M,

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The Journal of biological chemistry 2017 11 09() pii 10.1074/jbc.M117.798256

Abstract

HIV-1 reverse transcriptase (HIV-1 RT) possesses both DNA polymerase activity and RNase H activity that act in concert to convert single-stranded RNA of the viral genome to double-stranded DNA that is then integrated into the DNA of the infected cell. Reverse transcriptase-catalyzed reverse transcription critically relies on the proper generation of a polypurine tract (PPT) primer. However, the mechanism of PPT primer generation and the features of the PPT sequence that are critical for its recognition by HIV-1 RT remain unclear. Here, we used a chemical crosslinking method together with MD simulations and single-molecule assays to study the mechanism of PPT primer generation. We found that the PPT was specifically and properly recognized within covalently tethered HIV-1 RT-nucleic acid complexes. These findings indicated that recognition of the PPT occurs within a stable catalytic complex after its formation. We found that this unique recognition is based on two complementary elements that rely on the PPT sequence: RNase H sequence preference and incompatibility of the poly-rA/dT tract of the PPT with the nucleic acid conformation that is required for RNase H cleavage. The latter results from rigidity of the poly-rA/dT tract and leads to base-pair slippage of this sequence upon deformation into a catalytically relevant geometry. In summary, our results reveal an unexpected mechanism of PPT primer generation based on specific dynamic properties of the polyrA/dT segment and help advance our understanding of the mechanisms in viral RNA reverse transcription.

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