Insights into the multiple roles of pausing in HIV-1 reverse transcriptase-promoted strand transfers.
We previously analyzed the role of pausing induced by hairpin structures within RNA templates in facilitating strand transfer by HIV-1 RT (reverse transcriptase). We proposed a multistep transfer mechanism in which pause-induced RNase H cuts within the initial RNA template (donor) expose regions of cDNA. A second homologous RNA template (acceptor) can interact with the cDNA at such sites, initiating transfer. The acceptor-cDNA hybrid is thought to then propagate by branch-migration, eventually catching up with the primer terminus and completing the transfer. The prominent pause site in the template system facilitated acceptor invasion; however, very few of the transfers terminated at this pause. To examine the effects of homology on pause-promoted transfer, we increased template homology before the pause site, from 19 nucleotides (nt) in the initial template system to 52 nt in the new system. Significantly, the increased homology enhanced transfers 3-fold, with 32% of the transfers now terminating at the pause site. Additionally, the acceptor cleavage profile indicated the creation of a new invasion site in the added region of homology. NC (nucleocapsid) increased the strand transfer throughout the whole template. However, the prominent hot spot for internal transfer remained, which was still at the pause site. We interpret the new results to mean that pause sites can also serve to stall DNA synthesis, allowing acceptor invasions initiated earlier in the template to catch up with the primer terminus.
Gao
Lu
L
Department of Biochemistry and Biophysics, University of Rochester, New York 14642, USA.
Balakrishnan
Mini
M
Roques
Bernard P
BP
Bambara
Robert A
RA
D004247:DNA / Q000096:biosynthesis; D018076:DNA, Complementary / Q000378:metabolism*; D054303:HIV Reverse Transcriptase / Q000378:metabolism / Q000502:physiology*; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D012313:RNA / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013698:Templates, Genetic
D018076:DNA, Complementary; D012313:RNA; D004247:DNA; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H