TY - JOUR
T1 - Mechanism of polypurine tract primer generation by HIV-1 reverse transcriptase
AU - Figiel, Malgorzata
AU - Krepl, Miroslav
AU - Park, Sangwoo
AU - Poznański, Jaroslaw
AU - Skowronek, Krzysztof
AU - Golab, Agnieszka
AU - Ha, Taekjip
AU - Šponer, Jiří
AU - Nowotny, Marcin
N1 - Publisher Copyright:
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2018/1/5
Y1 - 2018/1/5
N2 - HIV-1 reverse transcriptase (RT) possesses both DNA polymerase activity and RNase H activity that act in concert to convert single-stranded RNA of the viral genome to doublestranded 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-1RTremain unclear. Here, we used a chemical cross-linking method together with molecular dynamics 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.Wefound that this unique recognition is based on two complementary elements that rely on the PPT sequence: RNaseHsequence preference and incompatibility of the poly(rA/dT) tract of the PPT with the nucleic acid conformation that is required for RNaseHcleavage. The latter results from rigidity of the poly(rA/dT) tract and leads to base-pair slippageof this sequenceupondeformation into a catalytically relevant geometry. In summary, our results reveal an unexpected mechanism of PPT primer generation based on specific dynamic properties of the poly(rA/dT) segment and help advance our understanding of the mechanisms in viral RNA reverse transcription.
AB - HIV-1 reverse transcriptase (RT) possesses both DNA polymerase activity and RNase H activity that act in concert to convert single-stranded RNA of the viral genome to doublestranded 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-1RTremain unclear. Here, we used a chemical cross-linking method together with molecular dynamics 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.Wefound that this unique recognition is based on two complementary elements that rely on the PPT sequence: RNaseHsequence preference and incompatibility of the poly(rA/dT) tract of the PPT with the nucleic acid conformation that is required for RNaseHcleavage. The latter results from rigidity of the poly(rA/dT) tract and leads to base-pair slippageof this sequenceupondeformation into a catalytically relevant geometry. In summary, our results reveal an unexpected mechanism of PPT primer generation based on specific dynamic properties of the poly(rA/dT) segment and help advance our understanding of the mechanisms in viral RNA reverse transcription.
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U2 - 10.1074/jbc.M117.798256
DO - 10.1074/jbc.M117.798256
M3 - Article
C2 - 29122886
AN - SCOPUS:85040114143
SN - 0021-9258
VL - 293
SP - 191
EP - 202
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 1
ER -