C-terminal region of bacterial Ku controls DNA bridging, DNA threading and recruitment of DNA ligase D for double strand breaks repair

Abstract

Non-homologous end joining is a ligation process repairing DNA double strand breaks in eukaryotes and many prokaryotes. The ring structured eukaryotic Ku binds DNA ends and recruits other factors which can access DNA ends through the threading of Ku inward the DNA, making this protein a key ingredient for the scaffolding of the NHEJ machinery. However, this threading ability seems unevenly conserved among bacterial Ku. As bacterial Ku differ mainly by their C-terminus, we evaluate the role of this region in the loading and the threading abilities of Bacillus subtilis Ku and the stimulation of the DNA ligase LigD. We identify two distinct sub-regions: a ubiquitous minimal C-terminal region and a frequent basic C-terminal extension. We show that truncation of one or both of these sub-regions in Bacillus subtilis Ku impairs the stimulation of the LigD end joining activity in vitro. We further demonstrate that the minimal C-terminus is required for the Ku-LigD interaction, whereas the basic extension controls the threading and DNA bridging abilities of Ku. We propose that the Ku basic C-terminal extension increases the concentration of Ku near DNA ends, favoring the recruitment of LigD at the break, thanks to the minimal C-terminal sub-region.

Multiple and Variable NHEJ-Like Genes Are Involved in Resistance to DNA Damage in Streptomyces ambofaciens

“…All these proteins harbor the conserved Ku core domain at their N-terminal side, the conserved minimal C-ter domain (red in Supplementary Figure S2), that has been shown to be required for the interaction with LigD in B. subtilis (McGovern et al, 2016), and a basic extended C-terminal domain, which is of 114, 128, and 47 aa, respectively. The pIs of these extended domains (11.64, 10.97, and 10.65) are consistent with those described for the majority of bacterial Ku proteins.…”

“…The pIs of these extended domains (11.64, 10.97, and 10.65) are consistent with those described for the majority of bacterial Ku proteins. This suggests that their roles in DNA binding as well as in the modulation of the ability to thread inward the DNA molecule are conserved in Streptomyces (Kushwaha and Grove, 2013; McGovern et al, 2016). Interestingly, a SAP domain, previously predicted in the S. coelicolor KuB homologue ( SCO0601 ) by Aravind and Koonin (2001) is also detected in S. ambofaciens KuB (at position 337–370, Supplementary Figure S2).…”

“…To date, characterized bacterial Ku are dimers. However, the elongated shape of the B. subtilis Ku (Ku Bsub ) dimer (McGovern et al, 2016) led to the elution of this protein with apparent molecular weight corresponding to a tetramer. Then KuA and KuB could also be dimers displaying elongated shape, as for Ku Bsub .…”

“…KuA, KuB, and KuC were able to protect a linear dsDNA molecule from the T5 exonuclease ( Figure 6A ), a property shared by the Ku Bsub protein (McGovern et al, 2016) and the Pseudomonas aeruginosa Ku against different exonucleases (Zhu and Shuman, 2010). This result suggests that these proteins are able to bind DNA ends.…”

“…LigD from Bacillus subtilis fused in C-terminal to a His 6 tag was purified as previously described McGovern et al (2016).…”