Expression and Partial Purification of Enzymatically Active Recombinant Ty1 Integrase in Saccharomyces Cerevisiae

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 1994 Mar 1

PMID 8127892

Citations 21

Authors

S P MOORE

D J Garfinkel

Affiliations

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Abstract

Integration of the Saccharomyces cerevisiae retrotransposon Ty1 into the genome requires Ty1 integrase (IN). Apparent functions of Ty1 IN are target-site determination, cleavage, and joining of donor strands. To further study the mechanism of Ty1 integration, an IN expression plasmid has been constructed for use in yeast. The recombinant IN coding sequence differs from mature Ty1 IN associated with Ty1 virus-like particles only in that it has several additional N-terminal amino acid codons. Inclusion of a polyhistidine tag facilitates purification of recombinant IN by metal chelate chromatography. Recombinant Ty1 IN is active in an in vitro assay with short double-stranded oligonucleotide substrates and has biochemical properties similar to those observed with Ty1 virus-like particles. The full-length Ty1 IN produced in yeast should be useful for further biochemical, genetic, and structural analyses of Ty1 integration and for comparative analyses with retroviral IN proteins.

Citing Articles

A proteomic screen of Ty1 integrase partners identifies the protein kinase CK2 as a regulator of Ty1 retrotransposition.

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Ty1 integrase is composed of an active N-terminal domain and a large disordered C-terminal module dispensable for its activity in vitro.

Nguyen P, Conesa C, Rabut E, Bragagnolo G, Gouzerh C, Fernandez-Tornero C J Biol Chem. 2021; 297(4):101093.

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The Ty1 LTR-retrotransposon of budding yeast, .

Curcio M, Lutz S, Lesage P Microbiol Spectr. 2015; 3(2):1-35.

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Ty1 integrase overexpression leads to integration of non-Ty1 DNA fragments into the genome of Saccharomyces cerevisiae.

Friedl A, Kiechle M, Maxeiner H, Schiestl R, Eckardt-Schupp F Mol Genet Genomics. 2010; 284(4):231-42.

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Functional analysis of N-terminal residues of ty1 integrase.

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