Insights into the functional coordination of LigD and Ku in bacterial nonhomologous end joining
摘要
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 (KuBs) and LigD (LigDBs). The ring-shaped KuBs homodimer binds DNA ends, promotes their synapsis, and recruits LigDBs. LigDBs 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 LigDBs 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 LigDBs catalyzes these steps without dissociating from DNA, being this processive catalysis dependent on KuBs-mediated synapsis. We further show that the joining ability of KuBs 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 KuBs-LigDBs interactions during NHEJ, providing new mechanistic insights into bacterial DSB repair.