Construction Of LigD And Ku Mutants In Mycobacterium Smegmatis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a microorganism that is among the greastest enemies’ of humanity. According to the World Health Organization’s prediction, one in three of the world’s population are infected with M. tuberculosis. And WHO data shows that in2011there are8.7million people fell ill with TB, including1.1million cases among people with HIV. An estimated1.4million people died from TB. M. tuberculosis coinfected with HIV cause large numbers of death. Even though several anti-mycobacterial drugs have been discovered and are currently in use, the results of the applied chemotherapy are far from satisfactory. TB is a problem that has never gone away.Double-strand break (DSB) is a kind of DNA damage which widespread in living creatures and it’s leathal to cells. DSBs are generally repaired by the pathway of homologous recombination or by DNA nonhomologous end-joining (NHEJ). NHEJ was first found in mammal cells, and had been considered only exist in eukaryote. In eukaryotic cells, repair of DNA double-strand breaks by the nonhomologous end-joining pathway is critical for genomic stability. However, following studies found NHEJ also generally exist in pathogenic microorganisms, with a relationship to drug resistant and immune evasion.Now we know that in Mycobacterial NHEJ is a two-component system, which contains LigD and Ku protein. And the function of these two proteins are well understood. While the regulation and interaction mechanism of LigD and Ku is not very clear. Recently our laboratory found a deacetylase which is called Sir2may participate in NHEJ pathway. And previous experiments indicate that M. smegmatis KuK29have εK-Ac modification. This suggests the relationship between the regulation of NHEJ and εK-Ac modification of KuK29.We use Mycobacterium smegmatis as a model organism to generate unmarked mutants of LigD and Ku by gene replacement. This mutants contains ligDΔPolDom, ku-His, kuR29and kuQ29. The progress of unmarked gene replacement involve two kinds of plasmid. The first one is p1NIL, it allows manipulation of the target gene sequence at a variety of convenient restriction sites. The second is pGOAL, it provides a marker gene cassette which is flanked by PacI restriction enzyme sites. The marker gene cassette contains an antibiotic resistance gene, lacZ and sacB. The final suicide plasmid vector is then obtained by cloning a marker cassette from a pGOAL vector into the single PacI site of the pNIL vector with the modified gene of interest. Finally, we get mutants after the screening of second cross-over.We found that the length of homologous arms, final concentration of alkali in plasmid treatment, pH of solution in precipitating DNA experiment and incubation time after the first cross-over all affect the construction of mutant strains. Our results suggest that His-tag in C-terminal of M. smegmatis Ku don’t influence the activity of NHEJ. This result eliminates the background effect caused by His-tag in NHEJ activity tests of M. smegmatis mutant ku. These mutants provide important materials for the research of NHEJ regulation.