Dynamics of Ku and bacterial non-homologous end-joining characterized using single DNA molecule analysis

Abstract

We use single-molecule techniques to characterize the dynamics of prokaryotic DNA repair by non-homologous end-joining (NHEJ), a system comprised only of the dimeric Ku and Ligase D (LigD). The Ku homodimer alone forms a ∼2 s synapsis between blunt DNA ends that is increased to ∼18 s upon addition of LigD, in a manner dependent on the C-terminal arms of Ku. The synapsis lifetime increases drastically for 4 nt complementary DNA overhangs, independently of the C-terminal arms of Ku. These observations are in contrast to human Ku, which is unable to bridge either of the two DNA substrates. We also demonstrate that bacterial Ku binds the DNA ends in a cooperative manner for synapsis initiation and remains stably bound at DNA junctions for several hours after ligation is completed, indicating that a system for removal of the proteins is active in vivo. Together these experiments shed light on the dynamics of bacterial NHEJ in DNA end recognition and processing. We speculate on the evolutionary similarities between bacterial and eukaryotic NHEJ and discuss how an increased understanding of bacterial NHEJ can open the door for future antibiotic therapies targeting this mechanism.

LigD: A Structural Guide to the Multi-Tool of Bacterial Non-Homologous End Joining

“…The resulting predictions of Ku shows that the shorter C-terminus of M. tuberculosis Ku may take on some structure, with alpha helices binding the C-terminus to the core, interspersed with disordered loops ( Figure 2C ). P. aeruginosa and B. subtilis Ku, though, have longer, disordered C-termini, corroborated by ColabFold’s low confidence score in positioning the modelled C-termini ( Figures 2D,E ) ( Jumper et al, 2021 ; Mirdita et al, 2021 ; Öz et al, 2021 ).…”

“…To achieve DNA DSB repair, prokaryotic NHEJ is dependent on the Ku70/80 homolog, Ku, and a multi-functional ATP-dependent ligase, LigD ( Figure 1 ) ( Weller et al, 2002 ; Bowater et al, 2006 ). Ku recognizes, binds to, and bridges across the ends of the double-strand break, protecting the DNA ends from further damage ( Shuman and Glickman, 2007 ; Öz et al, 2021 ). Ku homologs from B. subtilis and P.aeruginosa have reported lyase activity, useful for processing of the DSB ends, by removing abasic sites that may interfere with repair ( De Ory et al, 2014 ).…”

“…The minimal C-terminal region is conserved amongst bacteria, while the extended C-terminal region has low sequence conservation and is highly variable in length between organisms ( Figures 2A,B ) ( Kushwaha and Grove, 2013b ; McGovern et al, 2016 ). The core domain is predicted to form a ring-shaped structure that encircles DNA, much like eukaryotic Ku70/80, based on high sequence homology, and various in silico atomic structures from B. subtilis and M. tuberculosis ( McGovern et al, 2016 ; Jumper et al, 2021 ; Öz et al, 2021 ). The structure of the C-terminus, though, is likely variable.…”

Mechanistic Insights From Single-Molecule Studies of Repair of Double Strand Breaks

“…The subcomplex containing Ku and DNA-PKcs and stabilized by PAXX (∼2 s) appears consistent with the long range synapsis, while the full complex further stabilized by XRCC4-LIG4 and XLF (∼66 s) would correspond to the short range synapsis ( Stinson and Loparo, 2021 ). Most recently, the same technique was applied to demonstrate the dynamic properties of prokayrotic NHEJ synapsis involving just the Ku heterodimer and Ligase D ( Oz et al, 2021 ). Although debates remain regarding whether DNA-PKcs is required for synapsis, as the results appear to be dependent on the system employed, recent single-molecule work have unambiguously shown the process to be a dynamic process with distinct stages.…”

Editorial: Single-molecule studies of DNA–protein interactions collection 2021

“…Öz et al. ( 3 ) used a technique derived from magnetic tweezers, called molecular DNA forceps, where the ends of two DNA molecules are maintained in close proximity by a third DNA molecule that serves as a bridge. Using DNA forceps, they characterized the activity Ku and LigD from the bacteria Bacillus subtilis during non-homologous end-joining.…”