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Nucleic Acids Research, Vol 27, Issue 24 4679-4686, Copyright © 1999 by Oxford University Press

ARTICLES

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

S Yoo and WS Dynan
Program in Gene Regulation, Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912, USA.

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 DNA-PKcs makes direct contact with the DNA termini, occupying an approximately 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.
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