Abstract
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.
Highlights
Tivities are functionally separable [3,4,5,6], the polymerase and RNase H domains function in an interdependent manner [3, 7,8,9,10]
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 polypurine tract (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
The polymerase activity extends both RNA and DNA primers, efficient extension from RNA primers appears limited to the host cell-derived tRNA primer used for minus-strand DNA synthesis and the primer used for plussense DNA synthesis that is derived from the polypurine tract (PPT) sequence in the viral genome [12,13,14,15,16,17,18,19,20]
Summary
Tivities are functionally separable [3,4,5,6], the polymerase and RNase H domains function in an interdependent manner [3, 7,8,9,10]. Cleavage Analysis of RNA Oligonucleotide Primers—0.1 pmol of hybrid substrate containing 5Ј-end-labeled primer with or without a downstream oligonucleotide was incubated with 1 pmol of MMLV reverse transcriptase or RT⌬Pol in a 20-l reaction containing RNase H
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