Extension and cleavage of the polypurine tract plus-strand primer by Ty1 reverse transcriptase.
Using hybrid RNA/DNA substrates containing the polypurine tract (PPT) plus-strand primer, we have examined the interaction between the Ty1 reverse transcriptase (RT) and the plus-strand initiation complex. We show here that, although the PPT sequence is relatively resistant to RNase H cleavage, it can be cleaved internally by the polymerase-independent RNase H activity of Ty1 RT. Alternatively, this PPT can be used to initiate plus-strand DNA synthesis. We demonstrate that cleavage at the PPT/DNA junction occurs only after at least 9 nucleotides are extended. Cleavage leaves a nick between the RNA primer and the nascent plus-strand DNA. We show that Ty1 RT has a strand displacement activity beyond a gap but that the PPT is not efficiently re-utilized in vitro for another round of DNA synthesis after a first plus-strand DNA has been synthesized and cleaved at the PPT/U3 junction.
D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D017931:DNA Primers; D004261:DNA Replication; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011687:Purines / Q000737:chemistry*; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D018626:Retroelements / Q000235:genetics*; D016914:Ribonuclease H / Q000737:chemistry; D013698:Templates, Genetic
D017931:DNA Primers; D011687:Purines; C034477:RNA primers; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D018626:Retroelements; D012313:RNA; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H; C030985:purine