Expression of Mycobacterium tuberculosis Ku and Ligase D in Escherichia coli results in recA-independent DNA end-joining at regions of microhomology

Malyarchuk, Svitlana, Wright, Douglas, Castore, Reneau, Klepper, Emily, Weiss, Bernard, Doherty, Aidan J and Harrison, Lynn (2007) Expression of Mycobacterium tuberculosis Ku and Ligase D in Escherichia coli results in recA-independent DNA end-joining at regions of microhomology. DNA repair, 6 (10). pp. 1413-1424. ISSN 1568-7864

Full text not available from this repository.

Abstract

Unlike Escherichia coli, Mycobacterium tuberculosis (Mt) expresses a Ku-like protein and an ATP-dependent DNA ligase that can perform non-homologous end-joining (NHEJ). We have expressed the Mt-Ku and Mt-Ligase D in E. coli using an arabinose-inducible promoter and expression vectors that integrate into specific sites in the E. coli chromosome. E. coli strains have been generated that express the Mt-Ku and Mt-Ligase D on a genetic background that is wild-type for repair, or deficient in either the RecA or RecB protein. Transformation of these strains with linearized plasmid DNA containing a 2 bp overhang has demonstrated that expression of both the Mt-Ku and Mt-Ligase D is required for DNA end-joining and that loss of RecA does not prevent this double-strand break repair. Analysis of the re-joined plasmid has shown that repair is predominantly inaccurate and results in the deletion of sequences. Loss of RecB did not prevent the formation of large deletions, but did increase the amount of end-joining. Sequencing the junctions has revealed that the majority of the ligations occurred at regions of microhomology (1¿4 bps), eliminating one copy of the homologous sequence at the junction. The Mt-Ku and Mt-Ligase D can therefore function in E. coli to re-circularize linear plasmid.

Item Type: Article
Schools and Departments: School of Life Sciences > Sussex Centre for Genome Damage and Stability
Depositing User: Bernard Weiss
Date Deposited: 06 Feb 2012 19:46
Last Modified: 13 Jun 2012 13:56
URI: http://sro.sussex.ac.uk/id/eprint/22116
📧 Request an update