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 - Funding Information:
This work was initially supported by a grant from the Polish National Science Center (Contract N N301 439738); later by an International Early Career Scientist grant from the Howard Hughes Medical Institute (both to M. N.); and by a Foundation for Polish Science “Ideas for Poland” award (to M. N.), Czech Science Foundation Grant P305/12/G034 (to J. Sˇ. and M. K.), Ministry of Education, Youth and Sports of the Czech Republic Project LO1305 (to J. Sˇ.),PraemiumAcademiae(toJ. Sˇ.),andNationalInstitutesofHealthGrant GM065367 and United States National Science Foundation Grant PHY 1430124 (to S. P. and T. H.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the respon-sibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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 -