title: Specific cleavages by RNase H facilitate initiation of plus-strand RNA synthesis by Moloney murine leukemia virus.
abstract: Successful generation, extension, and removal of the plus-strand primer is integral to reverse transcription. For Moloney murine leukemia virus, primer removal at the RNA/DNA junction leaves the 3' terminus of the plus-strand primer abutting the downstream plus-strand DNA, but this 3' terminus is not efficiently reutilized for another round of extension. The RNase H cleavage to create the plus-strand primer might similarly result in the 3' terminus of this primer abutting downstream RNA, yet efficient initiation must occur to synthesize the plus-strand DNA. We hypothesized that displacement synthesis, RNase H activity, or both must participate to initiate plus-strand DNA synthesis. Using model hybrid substrates and RNase H-deficient reverse transcriptases, we found that displacement synthesis alone did not efficiently extend the plus-strand primer at a nick with downstream RNA. However, specific cleavage sites for RNase H were identified in the sequence immediately following the 3' end of the plus-strand primer. During generation of the plus-strand primer, cleavage at these sites generated a gap. When representative gaps separated the 3' terminus of the plus-strand primer from downstream RNA, primer extension significantly improved. The contribution of RNase H to the initiation of plus-strand DNA synthesis was confirmed by comparing the effects of downstream RNA versus DNA on plus-strand primer extension by wild-type reverse transcriptase. These data suggest a model in which efficient initiation of plus-strand synthesis requires the generation of a gap immediately following the plus-strand primer 3' terminus.
authors.affiliation: Department of Microbiology, School of Medicine, University of Washington, Seattle 98195-7242, USA.
authors.forename: Sharon J
authors.initials: SJ
authors.lastname: Schultz
authors.forename: Miaohua
authors.initials: M
authors.lastname: Zhang
authors.forename: James J
authors.initials: JJ
authors.lastname: Champoux
mesh_terms: D000818:Animals; D001483:Base Sequence; D004279:DNA, Viral / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics / Q000378:metabolism*; D012313:RNA; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D004279:DNA, Viral; C034477:RNA primers; D012367:RNA, Viral; D012313:RNA; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H