Geometry of a complex formed by double strand break repair proteins at a single DNA end: Recruitment of DNA-PKcs induces inward translocation of Ku protein

• Published in: Nucleic acids research Vol. 27; no. 24; pp. 4679 - 4686
• Main Authors: Yoo, Sunghan, Dynan, William S.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 15.12.1999; Oxford Publishing Limited (England)
• Subjects: Antigens, Nuclear; Base Sequence; Binding Sites; Cell Nucleus - metabolism; Cross-Linking Reagents; DNA - chemistry; DNA - metabolism; DNA Damage; DNA Helicases; DNA Probes - chemistry; DNA Repair; DNA-Activated Protein Kinase; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - metabolism; HeLa Cells; Humans; Kinetics; Ku Autoantigen; Ku protein; Models, Molecular; Nuclear Proteins - chemistry; Nuclear Proteins - metabolism; Nucleic Acid Conformation; Protein Structure, Quaternary; Protein-Serine-Threonine Kinases - chemistry; Protein-Serine-Threonine Kinases - metabolism
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/27.24.4679

Ku protein and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are essential components of the double-strand break repair machinery in higher eukaryotic cells. Ku protein binds to broken DNA ends and recruits DNA-PKcs to form an enzymatically active complex. To characterize the arrangement of proteins in this complex, we developed a set of photocross-linking probes, each with a single free end. We have previously used this approach to characterize the contacts in an initial Ku-DNA complex, and we have now applied the same technology to define the events that occur when Ku recruits DNA-PKcs. The new probes allow the binding of one molecule of Ku protein and one molecule of DNA-PKcs in a defined position and orientation. Photocross-linking reveals that DNAPKcs makes direct contact with the DNA termini, occupying an ∼10 bp region proximal to the free end. Characterization of the Ku protein cross-linking pattern in the presence and absence of DNA-PKcs suggests that Ku binds to form an initial complex at the DNA ends, and that recruitment of DNA-PKcs induces an inward translocation of this Ku molecule by about one helical turn. The presence of ATP had no effect on protein-DNA contacts, suggesting that neither DNA-PK-mediated phosphorylation nor a putative Ku helicase activity plays a role in modulating protein conformation under the conditions tested.