Analysis of plus-strand primer selection, removal, and reutilization by retroviral reverse transcriptases.
The ability of reverse transcriptase to generate, extend, and remove the primer derived from the polypurine tract (PPT) is vital for reverse transcription, since this process determines one of the ends required for integration of the viral DNA. Based on the ability of the RNase H activity of Moloney murine leukemia virus reverse transcriptase to cleave a long RNA/DNA hybrid containing the PPT, it appears that cleavages that could generate the plus-strand primer can occur by an internal cleavage mechanism without any positioning by an RNA 5'-end, and such cleavages may serve to minimize cleavage events within the PPT itself. If the PPT were to be cleaved inappropriately just upstream of the normal plus-strand origin site, the resulting 3'-ends would not be extended by reverse transcriptase. Extension of the PPT primer by at least 2 nucleotides is sufficient for recognition and correct cleavage by RNase H at the RNA-DNA junction to remove the primer. Specific removal of the PPT primer after polymerase extension deviates from the general observation that primer removal occurs by cleavage one nucleotide away from the RNA-DNA junction and suggests that the same PPT specificity determinants responsible for generation of the PPT primer also direct PPT primer removal. Once the PPT primer has been extended and removed from the nascent plus-strand DNA, reinitiation at the resulting plus-strand primer terminus does not occur, providing a mechanism to prevent the repeated initiation of plus strands.
Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195-7242, USA.
S J
SJ
Schultz
M
M
Zhang
C D
CD
Kelleher
J J
JJ
Champoux
D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology* / Q000235:genetics; D009692:Nucleic Acid Heteroduplexes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D009843:Oligoribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012313:RNA / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000235:genetics*
D009692:Nucleic Acid Heteroduplexes; D009843:Oligoribonucleotides; C034477:RNA primers; D011994:Recombinant Proteins; D012313:RNA; D004247:DNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H