Role of integrase in reverse transcription of the Saccharomyces cerevisiae retrotransposon Ty1.
Reverse transcriptase (RT) with its associated RNase H (RH) domain and integrase (IN) are key enzymes encoded by retroviruses and retrotransposons. Several studies have implied a functional role of the interaction between IN and RT during the replication of retroviral and retrotransposon genomes. In this study, IN deletion mutants were used to investigate the role of IN on the RT activity of the yeast Saccharomyces cerevisiae retrotransposon Ty1. We have identified two domains of Ty1 integrase which have effects on RT activity in vivo. The deletion of a domain spanning amino acid residues 233 to 520 of IN increases the exogenous specific activity of RT up to 20-fold, whereas the removal of a region rich in acidic amino acid residues between residues 521 and 607 decreases its activity. The last result complements our observation that an active recombinant RT protein can be obtained if a small acidic tail mimicking the acidic domain of IN is fused to the RT-RH domain. We suggest that interaction between these acidic amino acid residues of IN and a basic region of RT could be critical for the correct folding of RT and for the formation of an active conformation of the enzyme.
Wilhelm
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Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg, France. wilhelm@ibmc.u-strasbg.fr
Wilhelm
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D000595:Amino Acid Sequence; D024342:Amino Acids, Acidic / Q000737:chemistry; D001483:Base Sequence; D015153:Blotting, Western; D017353:Gene Deletion; D016681:Genome, Fungal; D019426:Integrases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018626:Retroelements / Q000235:genetics*; D048348:Reverse Transcription; D016914:Ribonuclease H / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D024342:Amino Acids, Acidic; D011993:Recombinant Fusion Proteins; D018626:Retroelements; D019426:Integrases; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H