Plus-strand priming by Moloney murine leukemia virus. The sequence features important for cleavage by RNase H.
The reverse transcriptase-associated RNase H activity is responsible for producing the plus-strand RNA primer during reverse transcription. The major plus-strand initiation site is located within a highly conserved polypurine tract (PPT), and initiation of DNA replication at this site is necessary for proper formation of the viral long terminal repeats (LTRs). We present here a compilation of PPT sequences from an evolutionarily diverse group of retroviruses and retrotransposons, which reveals that there is a high degree of sequence conservation at this site. Furthermore, we found previously that secondary plus-strand origins, identified in vitro, also show strong similarity to the PPT. Taken together, these data suggest that RNase H recognizes a specific sequence at the PPT as a signal to cleave the RNA at a precise location, producing a primer for the initiation of plus-DNA strands. We have analyzed the RNase H recognition sequence by producing a large number of single and double mutations within the PPT. Our findings suggest that no single residue in the +5 to -6 region (where the cleavage occurs between -1 and +1) is essential; mutations at these positions introduced heterogeneity at the cleavage site, but cleavage is still predominantly at the correct location. Furthermore, base-pairing is not required at the +1 position of the RNase H cleavage site, but a mismatched base-pair at the -1 position causes imprecision in the cleavage reaction. Interestingly, the A residue at position -7 seems to be critical in positioning the RNase H enzyme for correct cleavage. The preference of the enzyme for cleaving between G and A residues may play a minor role in determining the specificity.
Rattray
A J
AJ
Department of Microbiology, School of Medicine, University of Washington, Seattle 98195.
Champoux
J J
JJ
D001482:Base Composition; D001665:Binding Sites; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism*; D008979:Moloney murine leukemia virus / Q000378:metabolism*; D009154:Mutation; D009693:Nucleic Acid Hybridization; D011687:Purines / Q000378:metabolism; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D016914:Ribonuclease H
D004279:DNA, Viral; D011687:Purines; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase; D004722:Endoribonucleases; D016914:Ribonuclease H; C030985:purine