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Nucleic Acids Research, Vol 27, Issue 23 4547-4552, Copyright © 1999 by Oxford University Press

ARTICLES

A sequence immediately upstream of the plus-strand primer is essential for plus-strand DNA synthesis of the Saccharomyces cerevisiae Ty1 retrotransposon

M Wilhelm, T Heyman, M Boutabout and FX Wilhelm
Unite Propre de Recherche 9002 du Centre National de la Recherche Scientifique, Institut de Biologie Moleculaire et Cellulaire, 15 rue Rene Descartes, 67084 Strasbourg Cedex, France.

Priming of plus-strand DNA is a critical step in reverse transcription of retroviruses and retrotransposons. All retroelements use an RNase H- resistant oligoribonucleotide spanning a purine-rich sequence (the polypurine tract or PPT) to prime plus-strand DNA synthesis. Plus- strand DNA synthesis of the yeast Saccharomyces cerevisiae Ty1-H3 retrotransposon is initiated at two sites, PPT1 and PPT2, located at the upstream boundary of the 3'-long terminal repeat and near the middle of the pol gene in the integrase coding region. The two plus- strand primers have the same purine-rich sequence GGGTGGTA. This sequence is not sufficient by itself to generate a plus-strand origin since two identical sequences located upstream of PPT2 in the integrase coding region are not used efficiently as primers for plus-strand DNA synthesis. Thus, other factors must be involved in the formation of a specific plus-strand DNA primer. We show here that mutations upstream of the PPT in a highly conserved T-rich region severely alters plus- strand DNA priming of Ty1. Our results demonstrate the importance of sequences or structural elements upstream of the PPT for initiation of plus-strand DNA synthesis.
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