Photocrosslinking locates a binding site for the large subunit of human replication protein A to the damaged strand of cisplatin-modified DNA

The repair proteins XPA, XPC and replication protein A (RPA) have been impl icated in the primary recognition of damaged DNA sites during nucleotide ex cision repair. Detailed structural information on the binding of these prot eins to DNA lesions is however lacking, We have studied the binding of huma n RPA (hRPA) and hRPA-XPA-complexes to model oligonucleotides containing a single 1,3-d(GTG)-cisplatin-modification by photocrosslinking and electroph oretic mobility shift experiments, The 70 kDa subunit of hRPA can be crossl inked with high efficiency to cisplatin-modified DNA probes carrying 5-iodo -2'-deoxyuridin (5-IdU) as crosslinking chromophore. High efficiency crossl inking is dependent on the presence of the DNA lesion and occurs preferenti ally at its 5'-side. Examination of the crosslinking efficiency in dependen ce on the position of the 5-IdU chromophore indicates a specific positionin g of hRPA with respect to the platination site, When hRPA and XPA are both present mainly hRPA is crosslinked to the DNA, Our mobility shift experimen ts directly show the formation of a stable ternary complex of hRPA, XPA and the damaged DNA, The affinity of the XPA-hRPA complex to the damaged DNA i s increased by more than one order of magnitude as compared to hRPA alone.