title: Cellular dynamics of Ku: characterization and purification of Ku-eGFP.
abstract: Ku is a predominantly nuclear protein that functions as a DNA double-strand-break (DSB) binding protein and regulatory subunit of the DNA-dependent protein kinase (DNA-PK). DNA-PK is involved in synapsis and remodeling of broken DNA ends during nonhomologous end-joining (NHEJ) of DNA DSBs. It has also recently been demonstrated that Ku plays roles in cytoplasmic and membrane processes, namely: interaction with matrix metalloproteinase 9, acting as a co-receptor for parvoviral infection, and also interacting with cell polarity protein, Par3. We present a method for creating stable expression of Ku-eGFP in CHO cells and extend the procedure to purify Ku-eGFP for in vitro assaying. We demonstrated that Ku-eGFP localizes to the nucleus of HeLa cells upon microinjection into the cytoplasm as well as localizing to laser induced DNA damage. We also characterized the diffusional dynamics of Ku in the nucleus and in the cytoplasm using fluorescence correlation spectroscopy (FCS). The FCS data suggest that whereas the majority of Ku (70%) in the nucleus is mobile and freely diffusing, in a cellular context, there also exists a significant slow process fraction (30%). Strikingly, in the cytoplasm, this immobile/slow moving fraction is even more pronounced (45%).
authors.affiliation: Philips Research, High Tech Campus 11, 5656AE Eindhoven, The Netherlands. dennis.merkle@philips.com
authors.forename: Dennis
authors.initials: D
authors.lastname: Merkle
authors.forename: Dan
authors.initials: D
authors.lastname: Zheng
authors.forename: Thomas
authors.initials: T
authors.lastname: Ohrt
authors.forename: Karin
authors.initials: K
authors.lastname: Crell
authors.forename: Petra
authors.initials: P
authors.lastname: Schwille
mesh_terms: D000818:Animals; D034961:Antigens, Nuclear / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D019281:Dimerization; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D000072200:Ku Autoantigen; D010766:Phosphorylation
chemical_list: D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen