Mycobacterium tuberculosis Ku can bind to nuclear DNA damage and sensitize mammalian cells to bleomycin sulfate

Abstract

Radiotherapy and chemotherapy are effective cancer treatments due to their ability to generate DNA damage. The major lethal lesion is the DNA double-strand break (DSB). Human cells predominantly repair DSBs by non-homologous end joining (NHEJ), which requires Ku70, Ku80, DNA-PKcs, DNA ligase IV and accessory proteins. Repair is initiated by the binding of the Ku heterodimer at the ends of the DSB and this recruits DNA-PKcs, which initiates damage signaling and functions in repair. NHEJ also exists in certain types of bacteria that have dormant phases in their life cycle. The Mycobacterium tuberculosis Ku (Mt-Ku) resembles the DNA-binding domain of human Ku but does not have the N- and C-terminal domains of Ku70/80 that have been implicated in binding mammalian NHEJ repair proteins. The aim of this work was to determine whether Mt-Ku could be used as a tool to bind DSBs in mammalian cells and sensitize cells to DNA damage. We generated a fusion protein (KuEnls) of Mt-Ku, EGFP and a nuclear localization signal that is able to perform bacterial NHEJ and hence bind DSBs. Using transient transfection, we demonstrated that KuEnls is able to bind laser damage in the nucleus of Ku80-deficient cells within 10 sec and remains bound for up to 2 h. The Mt-Ku fusion protein was over-expressed in U2OS cells and this increased the sensitivity of the cells to bleomycin sulfate. Hydrogen peroxide and UV radiation do not predominantly produce DSBs and there was little or no change in sensitivity to these agents. Since in vitro studies were unable to detect binding of Mt-Ku to DNA-PKcs or human Ku70/80, this work suggests that KuEnls sensitizes cells by binding DSBs, preventing human NHEJ. This study indicates that blocking or decreasing the binding of human Ku to DSBs could be a method for enhancing existing cancer treatments.

Persistently bound Ku at DNA ends attenuates DNA end resection and homologous recombination

“…We have shown that a homolog of Ku70/80 from Mycobacterium tuberculosis (Mt-Ku) localizes to laser-generated DSBs in both human and hamster cells [20]. Mt-Ku is a 30-kDa protein, which forms a homodimer and is similar to Ku70/80 as it binds to dsDNA ends and mediates end-joining of DSBs [28–30].…”

“…After 60 minutes, roughly 80% of Ku80 had dissociated from the DSBs, whereas only 20% of the Mt-Ku had dissociated from the DSBs. We hypothesize that Mt-Ku is retained at DSBs in non-S phase cells because it is not able to interact with DNA-PKcs and thus cannot mediate NHEJ-mediated repair of DSBs [20]. In S phase, we hypothesize that the mechanism which mediates Ku’s dissociation from S phase cell fails to recognize Mt-Ku.…”

“…The data clearly shows that when DSBs are persistently bound by Mt-Ku, a marked decrease (14.45%) in HR-mediated DSB repair is observed when compared to the Ku deficient cells (32.05%). HR was higher in the cells complemented with Mt-Ku than human Ku80 which likely occurs because Mt-Ku does not interact with DNA-PKcs and thus cannot mediate NHEJ-mediated repair of the I-SceI induced DSB [20]. As complementation of CHO deficient cells with human Ku can mediate NHEJ, a percentage of the I-SceI induced DSBs are repaired via NHEJ and thus are not observed in our assay as being repaired and results in a smaller % of cells able to be repaired by HR [23].…”

“…Two open reading frame expression vectors encoding the Ku homolog from Mycobacterium tuberculosis (Mt-Ku) were used (at outlined in [20]). Mt-Ku does not localize to the nucleus in eukaryotic cells so the nuclear localization signal (NLS) from human Ku70 (amino acids 539–556 KVTKRKHDNEGSGSKRPK) was added to target the Mt-Ku protein to the nucleus.…”

Evidence for double-strand break mediated mitochondrial DNA replication in Saccharomyces cerevisiae

“…Because these terminal domains are required for communication with eukaryotic NHEJ proteins, expression of bKu in eukaryotic cells should, in theory, bind to DNA DSBs and prevent repair due to lack of communication between bKu and eukaryotic repair proteins. Indeed, we showed previously that nuclear-targeted Mycobacterium tuberculosis Ku (MtKu) binds to laser-induced DSBs and increases sensitivity of human cancer cells to bleomycin sulfate, a DSB inducing agent ( 30 ). Nuclear-targeted MtKu also attenuates homologous recombination in mammalian cells ( 31 ).…”