Requirements for strand transfer between internal regions of heteropolymer templates by human immunodeficiency virus reverse transcriptase.
We have examined the ability of the reverse transcriptase (RT) from human immunodeficiency virus (HIV) to carry out strand transfer synthesis (i.e., switching of the primer to a new template) from internal regions of natural-sequence RNA. A 142-nucleotide RNA template (donor) primed with a specific 20-nucleotide DNA oligonucleotide was used to initiate synthesis. DNA oligonucleotides with homology to internal regions of the donor were used as acceptors. In this system, HIV RT produced strand transfer products. An HIV RT having RNase H depleted to 3% of normal (HIV RTRD) catalyzed the transfer reaction inefficiently. An RNase H-minus deletion mutant of murine leukemia virus RT was unable to catalyze strand transfer. HIV RTRD, however, efficiently catalyzed transfer when Escherichia coli RNase H was included in the reactions, while the mutant murine leukemia virus RT was not efficiently complemented by the E. coli enzyme. Evidently, RNase H activity enhances, or is required for, internal strand transfer. Two acceptors homologous to 27-nucleotide regions of the donor, one offset from the other by 6 nucleotides, were tested. The offset eliminated a sequence homologous to a prevalent DNA synthesis pause site in the donor. Strand transfer to this acceptor was about 25% less efficient, suggesting that RT pausing can enhance strand transfer. When the deoxynucleoside triphosphates in the reactions were reduced from 50 to 0.2 microM, increasing RT pausing, the efficiency of strand transfer also increased. A model for RT-catalyzed strand transfer consistent with our results is presented.
DeStefano
J J
JJ
Department of Biochemistry, University of Rochester, New York 14642.
Mallaber
L M
LM
Rodriguez-Rodriguez
L
L
Fay
P J
PJ
Bambara
R A
RA
D001483:Base Sequence; D004279:DNA, Viral / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology; D006678:HIV / Q000201:enzymology* / Q000235:genetics; D009052:Leukemia Virus, Murine / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D016914:Ribonuclease H / Q000494:pharmacology; D013379:Substrate Specificity; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000187:drug effects
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D012367:RNA, Viral; D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H