The role of Mycobacterium tuberculosis Ku and ligase D in an Escherichia coli model of non-homologous end-joining

Non-homologous end joining (NHEJ) is the process where two ends of DNA are directly joined by using only the base-pairing information at the termini. NHEJ is a relatively recent discovery in prokaryotes and the only prokaryotes that have been found to perform NHEJ are those that have a dormant phase in their life cycle. One example is Mycobacterium tuberculosis. In this organism, Ku and Ligase D have been identified as required for NHEJ. Ku binds DNA ends and recruits Ligase D to the double strand break. Ligase D has three domains, a polymerase, nuclease, and ligase domain which allow for terminal processing and eventual ligation of the DNA ends. Due to the slow growth and technical disadvantages of working with M. tuberculosis , much of the work with Ku and Ligase D from this pathogenic organism has been performed in vitro and model cell systems to study NHEJ are limited. To develop a model system that is useful in identifying the essential components of these two NHEJ proteins, arabinose inducible expression vectors containing the coding regions for M. tuberculosis Ku and Ligase D were integrated into phage attachment sites within the genome of Escherichia coli, which does not possess the ability to perform NHEJ. Strains were generated that were 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 demonstrated that expression of both Ku and Ligase D is required for DNA end-joining and that the elimination of RecA or RecB does not prevent this double strand break repair. Analysis of the re-joined plasmid has shown that repair is predominately inaccurate and results in deletion of sequences. I hypothesized that the nuclease activity of Ligase D may be responsible for the deletions that are observed in this model. Strains were then generated that expressed Ku and mutant versions of Ligase D. Loss of the nuclease domain resulted in an increase in the amount of total and accurate repair, which provides evidence that the domain does have nuclease activity. Bacteria that contained the polymerase domain alone had no re-circularization activity. Deletion of the N-terminal polymerase domain from Ligase D resulted in a complete loss of accurate repair and a significant reduction in total repair. In vitro evidence previously showed that the polymerase domain is the major Ku binding site. The polymerase domain is approximately 300 amino acids in size. Fusion proteins of 26, 62, or 100 amino acids of the polymerase domain with the nuclease and ligase domain did not, however, result in an increase in repair activity, indicating that the majority of the polymerase domain is required for normal function and interaction with Ku. Interestingly, the ligase domain alone was able to re-circularize plasmid DNA, but only in a Ku-dependent manner. This reveals a potential second site for Ku-Ligase D interaction within the ligase domain. This work has increased the understanding of the interaction of Ku and Ligase D by examining the importance of the Ligase D domains during repair in bacteria.