A sequence immediately upstream of the plus-strand primer is essential for plus-strand DNA synthesis of the Saccharomyces cerevisiae Ty1 retrotransposon.
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.
Wilhelm
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M
Unité Propre de Recherche 9002 du Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg Cedex, France.
Heyman
T
T
Boutabout
M
M
Wilhelm
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FX
D001483:Base Sequence; D017931:DNA Primers; D004271:DNA, Fungal / Q000096:biosynthesis*; D009154:Mutation; D020218:Response Elements; D018626:Retroelements; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D017931:DNA Primers; D004271:DNA, Fungal; D018626:Retroelements