Photocross-linking of an oriented DNA repair complex - Ku bound at a single DNA end

Ku protein binds broken DNA ends, triggering a double-strand DNA break repa ir pathway, The spatial arrangement of the two Ku subunits in the initial K u-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 Ru protein to be constrai ned in position and orientation, relative to a single free DNA end. Results suggest that this complex is roughly symmetric and that both Ku subunits m ake contact with an approximately equal area of the DNA, The complex has a strongly preferred orientation, with Ku70-DNA backbone contacts located pro ximal and Ku80-DNA backbone contacts located distal to the free end. Ku70 a lso contacts functional groups in the major groove proximal to the free end . Ku80 apparently does not make major groove contacts. Results are consiste nt with a model where the Ku70 and Ku80 subunits contact the major and mino r grooves of DNA, respectively.