Polypurine tract primer generation and utilization by moloney murine leukemia virus reverse transcriptase

During reverse transcription, the RNase H activity of reverse transcriptase specifically cleaves the viral genome within the polypurine tract (PPT) to create the primer used for the initiation of plus-strand DNA synthesis and nonspecifically cleaves the viral genome to facilitate synthesis of plus-s trand DNA. To understand how primer length and sequence affect generation a nd utilization of the PPT, we employed short hybrid substrates containing o r lacking the PPT to evaluate cleavage, extension, and binding by reverse t ranscriptase. Substrates containing RNAs with the correct 3' end for initia tion of plus-strand synthesis were extended equally well by reverse transcr iptase, but primer length affected susceptibility to RNase H cleavage. RNA substrates with 3' ends extending beyond the plus strand initiation site we re extended poorly but were specifically cleaved to generate the correct 3' end for initiation of plus-strand synthesis, Substrates containing RNAs la cking the PPT were cleaved nonspecifically and extended inefficiently. Spec ific cleavages to generate the plus-strand primer and 5'-end-directed cleav ages were kinetically favored over cleavages that destroyed the PPT primer or degraded other short RNA fragments. The PPT was not intrinsically resist ant to cleavage by the isolated RNase H domain, and the isolated polymerase domain extended RNA primers containing the PPT sequence irrespective of th e primer 3' end. These results provide insights into how reverse transcript ase generates and selectively utilizes the PPT primer for initiation of plu s strand DNA synthesis.