Misincorporation by HIV-1 reverse transcriptase promotes recombination via strand transfer synthesis.
Genome heterogeneity in retroviruses derives from poor fidelity of the reverse transcriptase (RT) and recombination via RT-catalyzed strand transfer synthesis. RTs lack proofreading ability, and they proficiently extend primers with mismatched termini. Recombination reactions carried out in vitro are accompanied by a high frequency of base substitution errors, suggesting a relationship. Here we provide evidence that misincorporation during RNA-directed DNA synthesis promotes strand transfer recombination. Experiments involved measurement of DNA synthesis, RNase H-directed cleavage, and strand transfer synthesis from preformed mismatched primers on RNA templates by human immunodeficiency virus (HIV) RT in vitro. A significant pause in synthesis occurred from a G(primer). rA(template) mismatch compared to the synthesis from a correctly paired (T.A) primer. The misincorporation-induced pause allowed an unusually large area of RT-RNase H-directed cleavage of the template RNA beneath the primer. Strand transfer to an acceptor molecule with sequence identical to the template RNA was about 50% more efficient than if the primer had had a correctly paired terminus. Overall transfer was measured over a large region of homology. Assuming that enhanced transfer occurs primarily at the site of the mismatch, the actual increase in transfer at that site must have been 1-2 orders of magnitude. Inclusion of a different acceptor molecule with complete complementarity to the originally mismatched 3' primer terminus resulted in an additional 2-fold increase in strand transfer efficiency. Overall, these results suggest the mechanism by which misincorporation during minus strand DNA synthesis in retroviral replication would promote high frequency recombination.
Palaniappan
C
C
Department of Biochemistry, University of Rochester, Rochester, New York 14642, USA.
Wisniewski
M
M
Wu
W
W
Fay
P J
PJ
Bambara
R A
RA
D017931:DNA Primers / Q000378:metabolism; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D016350:Leucine Zippers; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D011995:Recombination, Genetic; D016914:Ribonuclease H / Q000378:metabolism; D013698:Templates, Genetic; D013997:Time Factors
D017931:DNA Primers; D004279:DNA, Viral; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H