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

Nucleotide excision repair (NER)1 is a versatile DNA repair pathway that exists to remove helix-distorting DNA lesions that would otherwise interfere with essential DNA-dependent processes, such as DNA replication and transcription. Together, more than 30 eukaryotic proteins are involved in four distinct steps that include initial damage recognition, cleavage of the damaged strand, displacement of the strand, and resynthesis and ligation, and the entire process has been reconstituted using purified proteins (1, 2). The initial step of DNA damage recognition can occur by either of two distinct mechanisms. The first couples NER to transcription, ensuring that the transcribed strand of genes is efficiently repaired (3). The second targets genomic DNA and achieves damage recognition through proteins that have a higher affinity for damaged DNA than for undamaged DNA. To date, several proteins have shown damage-specific binding, including the XPC–hHR23B complex (XPC–hHR23B), replication protein A (RPA), XPA, and DDB (4). Of these, only XPC–hHR23B is dispensable for transcription-coupled repair (TCR) in the presence of a stalled RNA polymerase II (5) but is specifically required for global genomic DNA repair (GGR) (6). The 144 kDa XPC protein is found in a heterotrimeric complex with hHR23B, the 44 kDa human homologue of the yeast protein Rad23 (7) and centrin 2, an 18 kDa centrosome component (8). However, XPC–hHR23B is sufficient for in vitro reconstitution of NER (9–11). Abundant evidence suggests that XPC–hHR23B is the first complex to recognize and bind to damaged DNA during GGR. For example, XPC–hHR23B is required for the earliest detectable open complex formation around a lesion at the beginning of NER (12, 13) and is the only NER protein with sufficient specificity and affinity to produce a distinct DNase footprint on DNA (14). A recent study used highly purified NER proteins and permanganate footprinting to confirm that XPC–hHR23B is the first complex to recognize damaged DNA and initiate the opening of nine bases of the helix around a site of damage (15). Although XPC–hHR23B is likely the initiator of GGR, the role of XPC in damage recognition has not been without controversy. Previous reports of the binding activity of XPC note that although XPC binds to a wide variety of lesions such as UV-induced photoproducts, cisplatin adducts, and AAF adducts, XPC also binds with high affinity to undamaged DNA (16). XPC–hHR23B is additionally able to bind with equal affinity to substrates containing bubbles with or without damage, although only the damaged substrates are able to be repaired (17). However, the preference of XPC–hHR23B for damaged versus undamaged DNA has been shown to increase up to 400-fold with the addition of undamaged competitor DNA (10). Notably, the large preference observed by XPC–hHR23B for damaged DNA in equilibrium binding experiments is consistent with either an increased rate of association or a decreased rate of dissociation from damaged DNA; however, these studies are unable to distinguish between these possibilities. In this report, we have examined the pre-steady state kinetics of binding of XPC–hHR23B to undamaged and damaged DNA using the intrinsic fluorescence of XPC–hHR23B and stopped-flow analysis to better understand the mechanism of damage-specific DNA binding. Equilibrium binding experiments were performed with the same DNA substrates to provide additional insight into the binding of XPC–hHR23B to damaged and undamaged DNA. The results revealed that damage-specific binding by XPC–hHR23B was mostly attributable to a dramatically increased kon for cisplatin and UV-damaged duplex DNA substrates compared to that for the undamaged duplex substrate, with very little difference in the dissociation rate. XPC–hHR23B displayed a 3-fold higher affinity for a 1,3 d(GpXpG) cisplatin adduct than for a 1,2 d(GpG) adduct, which was due to an increased rate of dissociation from the 1,2 d(GpG) adduct. Notably, although XPC–hHR23B binds with high affinity to single-stranded DNA and damaged duplex DNA, the presence of cisplatin damage on single-stranded DNA resulted in a decrease in the kon and an increase in the koff compared to those of undamaged single-stranded DNA, suggesting that XPC–hHR23B likely binds to the undamaged strand or adjacent to the adduct but not to the adduct itself. These data provide additional evidence that recognition and binding by XPC–hHR23B are predominantly a function of altered DNA structure.