Photocross-linking of an Oriented DNA Repair Complex

Abstract

Ku protein binds broken DNA ends, triggering a double-strand DNA break repair pathway. The spatial arrangement of the two Ku subunits in the initial Ku-DNA complex, when the Ku protein first approaches the broken DNA end, is not well defined. We have investigated the geometry of the complex using a novel set of photocross-linking probes that force Ku protein to be constrained in position and orientation, relative to a single free DNA end. Results suggest that this complex is roughly symmetric and that both Ku subunits make contact with an approximately equal area of the DNA. The complex has a strongly preferred orientation, with Ku70-DNA backbone contacts located proximal and Ku80-DNA backbone contacts located distal to the free end. Ku70 also contacts functional groups in the major groove proximal to the free end. Ku80 apparently does not make major groove contacts. Results are consistent with a model where the Ku70 and Ku80 subunits contact the major and minor grooves of DNA, respectively.