2000
DOI: 10.1042/0264-6021:3480337
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Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase‒RNase H domain exhibits polymerase and RNase H activities

Abstract: Replication of the Saccharomyces cerevisiae Ty1 retrotransposon requires a reverse transcriptase capable of synthesizing Ty1 DNA. The first description of an active form of a recombinant Ty1 enzyme with polymerase and RNase H activities is reported here. The Ty1 enzyme was expressed as a hexahistidine-tagged fusion protein in Escherichia coli to facilitate purification of the recombinant protein by metal-chelate chromatography. Catalytic activity of the recombinant protein was detected only when amino acid res… Show more

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Cited by 18 publications
(20 citation statements)
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“…The Ty1 reverse transcription reaction also occurs in the presence of Mn 2+ , although to a much lesser extent [135-137]. Ty1 RT contains a triad of aspartate residues in the polymerase active site that is conserved among RTs.…”
Section: Post-translational Steps In Retrotranspositionmentioning
confidence: 99%
“…The Ty1 reverse transcription reaction also occurs in the presence of Mn 2+ , although to a much lesser extent [135-137]. Ty1 RT contains a triad of aspartate residues in the polymerase active site that is conserved among RTs.…”
Section: Post-translational Steps In Retrotranspositionmentioning
confidence: 99%
“…There are few data available on the mechanisms of action of reverse transcriptases from LTR-retrotransposons, excepted those obtained in the course of studies performed on the yeast Ty1 and Ty3 reverse transcriptases (Cristofari et al, 1999;Lener et al, 2002;Wilhelm et al, 2000). We therefore aimed to elucidate the general characteristics of a reverse transcriptase encoded by an LTR-retrotransposon, ZAM, from Drosophila melanogaster (Leblanc et al, 1997).…”
Section: Introductionmentioning
confidence: 98%
“…However, the recent availability of recombinant forms of each enzyme (25,26) has permitted analysis of both their DNA polymerase and ribonuclease H (RNase H) functions, as well as the structure of cis -acting signals governing the initiation of both (−) and (+) strand DNA synthesis (2630). A study of Uzun and Gabriel (31) assessed the three conserved aspartic acid residues of the DNA polymerase catalytic center of Ty1 RT (Asp129, Asp210 and Asp211), indicating that, while the second aspartate of the Ty1 -Y-L-D-D- motif could be altered without affecting DNA polymerase activity, this mutation was lethal for transposition.…”
Section: Introductionmentioning
confidence: 99%