Insights into the functional coordination of LigD and Ku in bacterial nonhomologous end joining

Abstract

Abstract Nonhomologous end joining (NHEJ) is a DNA repair pathway that directly ligates double-strand breaks (DSBs) and operates without a homologous template. In Bacillus subtilis , this process involves two core proteins: the Ku homodimer (Ku Bs ) and LigD (LigD Bs ). The ring-shaped Ku Bs homodimer binds DNA ends, promotes their synapsis, and recruits LigD Bs . LigD Bs is a multifunctional enzyme featuring an N-terminal ligase domain (LigDom) and a C-terminal polymerization domain (PolDom). PolDom, in addition to ribonucleotide insertion has an AP lyase activity that processes abasic sites (AP) near recessed 5′ ends. Here, it is studied whether a single LigD Bs molecule can execute all three essential NHEJ catalytic steps, AP site incision, nucleotide insertion, and ligation in a coordinated manner when AP sites are present. The results indicate that LigD Bs catalyzes these steps without dissociating from DNA, being this processive catalysis dependent on Ku Bs -mediated synapsis. We further show that the joining ability of Ku Bs is critical for preventing intramolecular ligation of DNA ends, which otherwise would compromise DSB repair. Finally, the use of chimeric and truncated Ku variants revealed the specificity and dynamic nature of Ku Bs -LigD Bs interactions during NHEJ, providing new mechanistic insights into bacterial DSB repair.