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 residues encoded by the integrase gene were added to the N-terminus of the reverse transcriptase-RNase H domain. This suggests that the integrase domain could play a role in proper folding of reverse transcriptase. Several biochemical properties of the Ty1 enzyme were analysed, including the effect of MgCl2, NaCl, temperature and of the chain terminator dideoxy GTP on its polymerase activity. RNase H activity was examined by monitoring the cleavage of a RNA-DNA template-primer. Our results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 14-nucleotide RNA-DNA heteroduplex. The recombinant protein produced in E. coli should be useful for further biochemical and structural analyses and for a better understanding of the role of integrase in the activation of reverse transcriptase.
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June 2000
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Research Article|
May 23 2000
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 Available to Purchase
Marcelle WILHELM;
Marcelle WILHELM
1Unité 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
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Mansour BOUTABOUT;
Mansour BOUTABOUT
1Unité 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
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François-Xavier WILHELM
François-Xavier WILHELM
1
1To whom correspondence should be addressed (e-mail wilhelm@;ibmc.u-strasbg.fr).
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Publisher: Portland Press Ltd
Received:
January 13 2000
Revision Received:
March 01 2000
Accepted:
March 15 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 348 (2): 337–342.
Article history
Received:
January 13 2000
Revision Received:
March 01 2000
Accepted:
March 15 2000
Citation
Marcelle WILHELM, Mansour BOUTABOUT, François-Xavier WILHELM; 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. Biochem J 1 June 2000; 348 (2): 337–342. doi: https://doi.org/10.1042/bj3480337
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