A multistep damage recognition mechanism for global genomic nucleotide excision repair

A mammalian nucleotide excision repair (NER) factor, the XPC-HR23B complex, can specifically bind to certain DNA lesions and initiate the cell-free re pair reaction. Here we describe a detailed analysis of its binding specific ity using various DNA substrates, each containing a single defined lesion. A highly sensitive gel mobility shift assay revealed that XPC-HR23B specifi cally binds a small bubble structure with or without damaged bases, whereas dual incision takes place only when damage is present in the bubble. This is evidence that damage recognition for NER is accomplished through at leas t two steps; XPC-HR23B first binds to a site that has a DNA helix distortio n, and then the presence of injured bases is verified prior to dual incisio n. Cyclobutane pyrimidine dimers (CPDs) were hardly recognized by XPC-HR23B , suggesting that additional factors may be required for CPD recognition. A lthough the presence of mismatched bases opposite a CPD potentiated XPC-HR2 3B binding, probably due to enhancement of the helix distortion, cell-free excision of such compound lesions was much more efficient than expected fro m the observed affinity for XPC-HR23B. This also suggests that additional f actors and steps are required for the recognition of some types of lesions. A multistep mechanism of this sort may provide a molecular basis for ensur ing the high level of damage discrimination that is required for global gen omic NER.