Pre-steady-state binding of damaged DNA by XPC-hHR23B reveals a kinetic mechanism for damage discrimination

The XPC-hHR23B complex (XPC-hHR23B) is a heterodimeric protein required for the initial step of DNA damage recognition in the global nucleotide excision repair (NER) pathway. A strong preference of XPC-hHR23B for UV- and cisplatin-damaged DNA has previously been demonstrated using equilibrium binding assays. To better understand the molecular mechanism of damage recognition by XPC-hHR23B, we carried out the pre-steady-state kinetic analysis of the XPC-hHR23B-DNA interactions using a stopped-flow fluorescence assay. XPC-hHR23B displays a faster k(on) for cisplatin- and UV-damaged duplex DNA than for undamaged DNA, with additional, minor effects on the k(off) rates. XPC-hHR23B has a high affinity for undamaged single-stranded DNA compared to duplex DNA, which can be largely attributed to a high rate of association. However, cisplatin damage on single-stranded DNA reduced the overall level of binding by a factor of 7, with nearly equal contributions from changes to the k(on) and k(off) rates. Together, these results support a model for initial damage recognition by XPC-hHR23B that is dependent on structural changes in the DNA, and not adduct chemistry.