Characterization of the roles of the catalytic domains of Mycobacterium tuberculosis ligase D in Ku-dependent error-prone DNA end joining.
We previously established an Escherichia coli strain capable of re-circularizing linear plasmid DNA by expressing the Mycobacterium tuberculosis Ku (Mt-Ku) and Mycobacterium tuberculosis ligase D (Mt-LigD) proteins from the E.coli chromosome. Repair was predominately mutagenic due to deletions at the termini. We hypothesized that these deletions could be due to a nuclease activity of Mt-LigD that was previously detected in vitro. Mt-LigD has three domains: an N-terminal polymerase domain (PolDom), a central domain with 3'-phosphoesterase and nuclease activity and a C-terminal ligase domain (LigDom). We generated bacterial strains expressing Mt-Ku and mutant versions of Mt-LigD. Plasmid re-circularization experiments in bacteria showed that the PolDom alone had no re-circularization activity. However, an increase in the total and accurate repair was found when the central domain was deleted. This provides further evidence that this central domain does have nuclease activity that can generate deletions during repair. Deletion of only the PolDom of Mt-LigD resulted in a complete loss of accurate repair and a significant reduction in total repair. This is in agreement with published in vitro work indicating that the PolDom is the major Mt-Ku-binding site. Interestingly, the LigDom alone was able to re-circularize plasmid DNA but only in an Mt-Ku-dependent manner, suggesting a potential second site for Ku-LigD interaction. This work has increased our understanding of the mutagenic repair by Mt-Ku and Mt-LigD and has extended the in vitro biochemical experiments by examining the importance of the Mt-LigD domains during repair in bacteria.
Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center at Shreveport, 1501 Kings Highway, Shreveport, LA 71130, USA.
Douglas
D
Wright
Austin
A
DeBeaux
Runhua
R
Shi
Aidan J
AJ
Doherty
Lynn
L
Harrison
D034961:Antigens, Nuclear / Q000378:metabolism*; D015153:Blotting, Western; D020134:Catalytic Domain; D053903:DNA Breaks, Double-Stranded; D011088:DNA Ligases / Q000737:chemistry* / Q000378:metabolism*; D004260:DNA Repair; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000072200:Ku Autoantigen; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D017384:Sequence Deletion / Q000235:genetics; D013329:Structure-Activity Relationship
D034961:Antigens, Nuclear; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D000072200:Ku Autoantigen; D011088:DNA Ligases; C493173:DNA ligase D, Mycobacterium tuberculosis