Pausing by retroviral DNA polymerases promotes strand transfer from internal regions of RNA donor templates to homopolymeric acceptor templates.
We have examined the ability of reverse transcriptases (RT) to catalyze strand transfer from internal regions of RNA templates, resulting in switching of a primer from one template to another. To study this phenomenon, we employed a system of donor and acceptor templates in which homologous strand transfer can occur from a homopolymeric sequence, positioned internally on the donor template. Our results indicate that reverse transcriptases from human immunodeficiency virus (HIV), avian myeloblastosis virus (AMV), and murine leukemia virus (MuLV) are all able to catalyze strand transfer from this sequence. Catalysis of this reaction is not dependent upon ribonuclease H (RNase H) activity, since an RNase H-deficient form of HIV-RT is able to catalyze the reaction efficiently. Additionally, N-ethylmaleimide, which inhibits RNase H but not polymerase activity, did not inhibit the template switching by either the native or RNase H-deficient forms of HIV-RT. Our data further indicate that template switching may be promoted by RT pausing at a specific site on the donor template. Conditions that increase RT pausing at this site also increase template switching. These results suggest that transient RT pausing at specific sites on the viral genome during reverse transcription may promote template switches that in turn lead to recombination.
Buiser
R G
RG
Department of Biochemistry, University of Rochester, NY 14642.
Bambara
R A
RA
Fay
P J
PJ
D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D016914:Ribonuclease H; D013698:Templates, Genetic
D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H