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Updated: Aug 31, 2026

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo
Published on: October 31, 2014
Extension and cleavage of the polypurine tract plus-strand primer by Ty1 reverse transcriptase
François-Xavier Wilhelm1, Marcelle Wilhelm, Abram Gabriel
1Institut de Biologie Moléculaire et Cellulaire, 15, rue R. Descartes, 67084 Strasbourg, France. wilhelm@ibmc.u-strasbg.fr
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.
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