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Expression of Mycobacterium tuberculosis Ku and Ligase D in Escherichia coli results in recA-independent DNA end-joining at regions of microhomology
journal contribution
posted on 2023-06-07, 23:58 authored by Svitlana Malyarchuk, Douglas Wright, Reneau Castore, Emily Klepper, Bernard Weiss, Aidan DohertyAidan Doherty, Lynn HarrisonUnlike 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.
History
Publication status
- Published
Journal
DNA repairISSN
1568-7864Publisher
ElsevierExternal DOI
Issue
10Volume
6Page range
1413-1424Pages
12.0Department affiliated with
- Sussex Centre for Genome Damage Stability Publications
Full text available
- No
Peer reviewed?
- Yes
Legacy Posted Date
2012-02-06Usage metrics
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