Abstract
To better understand the mechanism by which Ty1 RNase H creates the polypurine tract (PPT) primer, we have demonstrated the polymerase-dependent hydrolytic activity of Ty1 reverse transcriptase (RT) during minus-strand synthesis. Using RNase H and polymerase mutants of the recombinant Ty1 RT protein, we show that the two domains of Ty1 RT can act independently of one another. Our results indicate that RNA/DNA substrates containing a short RNA PPT, which serve as primers for plus-strand DNA synthesis, are relatively resistant to RNase H cleavage. RNA substrates with a correct 5' end but with 3' end extending beyond the plus-strand initiation site were cleaved specifically to generate the correct 3' end of the PPT. Using long RNA/DNA duplexes containing the PPT, we show that Ty1 RT is able to make specific internal cleavages that could generate the plus-strand primer with correct 5' and 3' ends. Long RNA/DNA duplexes with mutations in the PPT or in a U-rich region upstream of the PPT, which abolish plus-strand initiation in vivo, were not cleaved specifically at the 5' end of the PPT. Our work demonstrates that the in vitro enzyme can recapitulate key processes that control proper replication in vivo.
Highlights
Reverse transcriptase (RT)1 of retroviruses and LTR retrotransposons is a multifunctional enzyme that possesses RNAdependent and DNA-dependent DNA polymerase activities as well as RNase H activity that degrades the RNA strand of RNA/DNA duplexes
Our results indicate that RNA/DNA substrates containing a short RNA polypurine tracts (PPT), which serve as primers for plus-strand DNA synthesis, are relatively resistant to RNase H cleavage
Using HIV-1 reverse transcriptase (RT), two modes of RNase H activity can be distinguished: (i) a polymerase-dependent mode, which accompanies DNA synthesis and is positioned by the polymerase domain binding to the recessed nascent DNA 3Ј end; and (ii) a polymerase-independent mode, which occurs without DNA synthesis but is oriented by the polymerase domain binding to the recessed 5Ј end of RNA fragments paired to the DNA
Summary
100a 104 Ͻ3.2 a 100% activity equals 3.72 pmol of dGTP incorporated/g of microgram enzyme/h. In this study we have used hybrid RNA/DNA substrates containing the PPT sequence to evaluate how recombinant Ty1 RT extends PPT-containing primers during initiation of plusstrand DNA synthesis and cleaves RNA/DNA duplexes near the site of initiation of plus-strand DNA synthesis. By working with a genetically tractable endogenous retroelement, we can gain insights into the mechanism by which RNase H activities of RTs generate a PPT primer
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