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J. Biol. Chem., Vol. 278, Issue 48, 47678-47684, November 28, 2003

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

François-Xavier Wilhelm, Marcelle Wilhelm, and Abram Gabriel¶||

From the Institut de Biologie Moléculaire et Cellulaire, 15, rue R. Descartes, 67084 Strasbourg, France and the ^¶Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854

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.

Received for publication, May 16, 2003 , and in revised form, September 17, 2003.

^* This work was supported in part by Grant 9589 from the Association pour la Recherche contre le Cancer, by a CNRS grant (Poste DRA 4935) (to A. G.), and by National Institutes of Health Grant GM60534. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence may be addressed: IBMC, 15 rue R. Descartes, 67084 Strasbourg, France. Tel.: 33-3-88417006; Fax: 33-3-602218; E-mail: wilhelm{at}ibmc.u-strasbg.fr. ||To whom correspondence may be addressed: Dept. of Molecular Biology and Biochemistry, Rutgers University, CABM 306, 679 Hoes Lane, Piscataway, NJ 08854. Tel.: 732-235-5097; Fax: 732-235-4880; E-mail: gabriel{at}cabm.rutgers.edu.

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