DNA Conformational Distribution and Dynamics during Search and Recognition of Damaged Sites by DNA Repair Protein RAD4/XPC

Abstract

DNA damage repair proteins need to scan and identify DNA lesions in genomic DNA. In mammalian nucleotide excision repair (NER), lesion recognition is carried out by the Xeroderma Pigmentosum C protein complex (XPC). Rad4, a yeast XPC ortholog, recognizes DNA lesions by inserting a β-hairpin and flipping out damaged nucleotides away from the protein. Rad4, covalently tethered to undamaged DNA, flips out even normal nucleotides to form the same ‘recognition’ structure. Thus, lesion discrimination must depend on differences in the kinetics of recognition rather than structural differences in the stable states of the bound complexes. In this ‘kinetic gating’ mechanism, the probability that a freely diffusing Rad4 flips out nucleotides competes kinetically with Rad4 diffusing away from that site. Using laser temperature-jump spectroscopy, we have measured Rad4-induced nucleotide-flipping rates from within a 3-bp mismatch (model) lesion, for both freely diffusing and tethered Rad4, using 2-aminopurine (2AP) as a probe. Our results reveal 2-5 fold slower relaxation rates for 2AP-monitored nucleotide-flipping dynamics when Rad4 is tethered at the mismatch site compared to when it is freely diffusing, consistent with the notion of competing pathways if the protein can diffuse away from a lesion site. Additionally, we have combined the unique sensitivity of picoseconds-resolved fluorescence lifetime measurements with novel tricyclic cytosine analogs incorporated in DNA (FRET pair: tCo-tCnitro) as probes of DNA twisting/unwinding fluctuations. While the mismatched and matched DNA exhibited similar average FRET, suggesting similar average DNA conformations, the lifetime data reveal multiple distinct conformations for mismatched DNA in contrast to the single dominant conformation seen for matched DNA. These results indicate enhanced unwinding fluctuations in the vicinity of a mismatch “lesion”, perhaps signaling “distress” to a protein searching for damaged sites.