Expression of Mycobacterium tuberculosis Ku and Ligase D in Escherichia coli results in RecA and RecB-independent DNA end-joining at regions of microhomology.
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 2bp 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 (1-4bps), 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.
Malyarchuk
Svitlana
S
Department of Molecular and Cellular Physiology, Louisiana Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130, USA.
Wright
Douglas
D
Castore
Reneau
R
Klepper
Emily
E
Weiss
Bernard
B
Doherty
Aidan J
AJ
Harrison
Lynn
L
D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D000072200:Ku Autoantigen; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000378:metabolism*; D011938:Rec A Recombinases / Q000378:metabolism*
D034961:Antigens, Nuclear; D017931:DNA Primers; D004268:DNA-Binding Proteins; D011938:Rec A Recombinases; D000072200:Ku Autoantigen; D011088:DNA Ligases; C493173:DNA ligase D, Mycobacterium tuberculosis