Incomplete removal of the RNA primer for minus-strand DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase.
A synthetic RNA oligonucleotide (15-mer) corresponding to the 3' end of the lysine tRNA primer was hybridized to single-stranded DNA containing the human immunodeficiency virus type 1 (HIV-1) primer-binding site and extended with a DNA polymerase. The resulting structures were used to study primer removal by the RNase H activity of HIV-1 reverse transcriptase. The initial cleavage event removes the RNA primer as a 14-mer and leaves a single ribonucleotide A residue bound to the 5' end of the DNA strand. This result explains the observation by several groups that HIV-1 circle junctions contain 4 bp that are not present in the integrated provirus instead of the predicted 3 bp. Subsequent cleavage events occur at other sites internal to the RNA molecule, and the ribonucleotide A residue on the end of the DNA strand is ultimately removed. Therefore, the biologically relevant cleavage that produces the 14-mer reflects the kinetics of the reaction as well as a specificity for nucleic acid sequence. When the RNA oligonucleotide alone was hybridized to the primer-binding site and tested as a substrate for HIV-1 RNase H, the cleavage pattern near the 3' end of the RNA was altered.
Department of Microbiology, School of Medicine, University of Washington, Seattle 98195.
K A
KA
Pullen
L K
LK
Ishimoto
J J
JJ
Champoux
D001483:Base Sequence; D004279:DNA, Viral / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D012343:RNA, Transfer / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D012265:Ribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D013698:Templates, Genetic
D004279:DNA, Viral; D012367:RNA, Viral; D012265:Ribonucleotides; D012343:RNA, Transfer; D012194:RNA-Directed DNA Polymerase