In silicoandin vitrocharacterization of the mycobacterial protein Ku to unravel its role in non-homologous end-joining DNA repair

Abstract

Non–homologous end–joining DNA repair is essential for the survival and sustenance ofM·tuberculosis(Mtb) in the dormant stage of its life cycle. The ability of Mtb to sustain itself in the inactive form has been reported to be the critical factor for its resilience over the years. To unravel one of the salient features of the Mtb′s arsenal, we exploitedin silicoandin vitrotools to characterize the DNA binding properties of mycobacterial protein Ku (mKu) and its role in mycobacterial NHEJ. Here, we report the strong affinity of mKu for linear dsDNA exhibiting positive cooperativity for dsDNAs (40bp long or more). Molecular dynamics complemented within vitroexperiments showed that the DNA binding of mKu provides stability to both mKu homodimer and the DNA. Furthermore, mKu end capping of DNA was seen to protect the DNA termini against nucleolytic degradation by exonuclease. The DNA–mKu association formed higher–order oligomers probably due to the lodgement of two DNA molecules at opposite ends of the mKu homodimer. The ability of mKu to form continuous filament–like structures with DNA indicated its potential role in mycobacterial NHEJ synapsis.