Extension and Cleavage of the Polypurine Tract Plus-strand Primer by Ty1 Reverse Transcriptase

François-Xavier Wilhelm,Marcelle Wilhelm,Abram Gabriel

doi:10.1074/jbc.m305162200

François-Xavier Wilhelm, Marcelle Wilhelm + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m305162200

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Abstract

Using hybrid RNA/DNA substrates containing the polypurine tract (PPT) plus-strand primer, we have examined the interaction between the Ty1 reverse transcriptase (RT) and the plus-strand initiation complex. We show here that, although the PPT sequence is relatively resistant to RNase H cleavage, it can be cleaved internally by the polymerase-independent RNase H activity of Ty1 RT. Alternatively, this PPT can be used to initiate plus-strand DNA synthesis. We demonstrate that cleavage at the PPT/DNA junction occurs only after at least 9 nucleotides are extended. Cleavage leaves a nick between the RNA primer and the nascent plus-strand DNA. We show that Ty1 RT has a strand displacement activity beyond a gap but that the PPT is not efficiently re-utilized in vitro for another round of DNA synthesis after a first plus-strand DNA has been synthesized and cleaved at the PPT/U3 junction.

Highlights

During reverse transcription of the plus-strand RNA genome of long terminal repeat retroelements, the RNase H activity of reverse transcriptase (RT)1 degrades the genomic RNA while the polymerase activity synthesizes minus-strand DNA (Fig. 1) [1,2,3,4,5,6,7]
We have previously shown that the 9-nt polypurine tract (PPT) primer is relatively resistant to RNase H cleavage when this hybrid substrate is incubated with Ty1 RT [11]
Using RNA/DNA duplexes containing the PPT, we previously showed that a recombinant Ty1 RT is able to make specific internal cleavages that could generate the plusstrand primer with correct 5Ј and 3Ј ends

Summary

Introduction

During reverse transcription of the plus-strand RNA genome of long terminal repeat retroelements, the RNase H activity of reverse transcriptase (RT)1 degrades the genomic RNA while the polymerase activity synthesizes minus-strand DNA (Fig. 1) [1,2,3,4,5,6,7]. No 9R/25D run-off band was observed (Fig. 3, lanes 1 and 2), indicating the marked propensity of cleavage of the primer RNA by RNase H.

Results

Conclusion