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

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.