title: The development and applications of nonradioactive plate-formatted DNA-binding assay for Ku70/80, a multifunctional DNA-binding protein complex.
abstract: Ku is a heterodimer composed of p70 and p80, and is the regulatory subunit of DNA-dependent protein kinase. As a multifunctional DNA-binding protein complex, Ku plays important roles in DNA damage repair through non-homologous end joining and in V(D)J recombination. In addition, Ku has also been implicated in various biological functions including growth control, cell proliferation, cell cycle, chromosome maintenance, transcriptional regulation, apoptosis, and viral infection. In particular, using our Inverse Genomics (Immusol, Inc., San Diego, CA) platform technology, we recently identified Ku80 as an essential co-factor for human immunodeficiency virus replication. Although Ku has been studied extensively in the past years, its in-depth study as well as development as a drug target has been limited by conventional DNA-binding activity assay. Here we describe the development and applications of a nonradioactive DNA binding assay in the 96-well format. We show that this plate-formatted assay is more sensitive and allows for direct quantification when compared with an electrophoretic mobility shift assay. The establishment of this assay will not only facilitate structure and function studies on Ku, but also help the development of Ku protein or its DNA repair enzyme complex as a drug target.
authors.affiliation: Immusol, Inc., 10790 Roselle Street, San Diego, CA 92121, USA. ma@immusol.com
authors.forename: Hongwen
authors.initials: H
authors.lastname: Ma
authors.forename: Joseph
authors.initials: J
authors.lastname: Thibault
authors.forename: Yali
authors.initials: Y
authors.lastname: Lu
authors.forename: Carson
authors.initials: C
authors.lastname: Whiting
authors.forename: Shinong
authors.initials: S
authors.lastname: Long
authors.forename: Glen
authors.initials: G
authors.lastname: Lindwall
authors.forename: Krista
authors.initials: K
authors.lastname: Bennett
authors.forename: Lynn
authors.initials: L
authors.lastname: Truong
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Aimes
authors.forename: Flossie
authors.initials: F
authors.lastname: Wong-Staal
mesh_terms: D000818:Animals; D034961:Antigens, Nuclear / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005990:Glycerol / Q000494:pharmacology; D006801:Humans; D007313:Insecta; D000072200:Ku Autoantigen; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013678:Technology, Pharmaceutical / Q000379:methods*
chemical_list: D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D012965:Sodium Chloride; D004247:DNA; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D005990:Glycerol