Abstract
The mycobacterial nonhomologous end-joining pathway (NHEJ) involved in double-strand break (DSB) repair consists of the multifunctional ATP-dependent ligase LigD and the DNA bridging protein Ku. The other ATP-dependent ligases LigC and AEP-primase PrimC are considered as backup in this process. The engagement of LigD, LigC, and PrimC in the base excision repair (BER) process in mycobacteria has also been postulated. Here, we evaluated the sensitivity of Mycolicibacterium smegmatis mutants defective in the synthesis of Ku, Ku-LigD, and LigC1-LigC2-PrimC, as well as mutants deprived of all these proteins to oxidative and nitrosative stresses, with the most prominent effect observed in mutants defective in the synthesis of Ku protein. Mutants defective in the synthesis of LigD or PrimC/LigC presented a lower frequency of spontaneous mutations than the wild-type strain or the strain defective in the synthesis of Ku protein. As identified by whole-genome sequencing, the most frequent substitutions in all investigated strains were T→G and A→C. Double substitutions, as well as insertions of T or CG, were exclusively identified in the strains carrying functional Ku and LigD proteins. On the other hand, the inactivation of Ku/LigD increased the efficiency of the deletion of G in the mutant strain.
Highlights
Introduction published maps and institutional affilMycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a leading bacterial pathogen claiming 1.5 million lives each year [1]
nonhomologous end-joining pathway (NHEJ) Proteins Promote the Resistance of Mycobacteria to DNA Methylation and Ionizing radiation and desiccation cause single- and double-stranded breaks in the DNA, which must be repaired by homologous recombination (HR) or NHEJ
It was already reported that NHEJ-deficient strains (∆ku, ∆ligD, and ∆ku-ligD) of M. smegmatis are sensitive to ionizing radiation (IR) during the stationary phase and that NHEJ protects mycobacteria against the harmful effects of desiccation [13]
Summary
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a leading bacterial pathogen claiming 1.5 million lives each year [1]. Mtb is an intracellular pathogen, and its life cycle includes long states of persistence. As well as other pathogens, face a variety of harmful conditions during infection, caused by host defense mechanisms and drug treatments. As a very successful pathogen with outstanding adaptive properties, Mtb has developed a plethora of sophisticated mechanisms to subvert the host defense and to effectively enter and replicate in the harmful environment inside professional phagocytes, namely macrophages. Tubercle bacilli colonizing the mucosal surface of the lower respiratory epithelium are phagocytized by alveolar macrophages and exposed to various DNA-damaging assaults affecting the genome integrity, including reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI). ROS and RNI are responsible for the oxidation or alkylation of bases, covalent linking of two bases, or elimination of iations
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