Plus-strand priming by Moloney murine leukemia virus. The sequence features important for cleavage by RNase H.

Journal of Molecular Biology
A J Rattray, J J Champoux

Abstract

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...Continue Reading

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