title: 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.
authors.affiliation: Department of Molecular and Cellular Physiology, LSUHSC-S, Shreveport, LA, USA.
authors.forename: Reneau
authors.initials: R
authors.lastname: Castore
authors.forename: Cameron
authors.initials: C
authors.lastname: Hughes
authors.forename: Austin
authors.initials: A
authors.lastname: Debeaux
authors.forename: Jingxin
authors.initials: J
authors.lastname: Sun
authors.forename: Cailing
authors.initials: C
authors.lastname: Zeng
authors.forename: Shih-Ya
authors.initials: SY
authors.lastname: Wang
authors.forename: Kelly
authors.initials: K
authors.lastname: Tatchell
authors.forename: Runhua
authors.initials: R
authors.lastname: Shi
authors.forename: Kyung-Jong
authors.initials: KJ
authors.lastname: Lee
authors.forename: David J
authors.initials: DJ
authors.lastname: Chen
authors.forename: Lynn
authors.initials: L
authors.lastname: Harrison
mesh_terms: D000818:Animals; D034961:Antigens, Nuclear / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001761:Bleomycin / Q000494:pharmacology*; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004249:DNA Damage; D059766:DNA End-Joining Repair / Q000187:drug effects; D011088:DNA Ligases / Q000378:metabolism; D004270:DNA, Circular / Q000378:metabolism; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D000072200:Ku Autoantigen; D008322:Mammals; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D034961:Antigens, Nuclear; D001426:Bacterial Proteins; D004270:DNA, Circular; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D001761:Bleomycin; D049452:Green Fluorescent Proteins; D051747:DNA-Activated Protein Kinase; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D011088:DNA Ligases