Dynamics of DNA Damage Recognition by Nucleotide Excision Repair Protein XPC

Abstract

The first step in DNA damage repair is the recognition of and binding to the damaged site by proteins that scan the genome for lesions. In the nucleotide excision repair (NER) pathway, this initial damage recognition step is carried out by the Xeroderma Pigmentosum C protein (XPC). High resolution crystal structures of XPC bound to DNA containing one such lesion site show a series of conformational changes in the complex: (1) DNA is bent and unwound at the lesion site, (2) both the undamaged and damaged bases are flipped out, and (3) the bent/flipped-out structure is stabilized by the insertion of a Beta-hairpin of XPC. However, in what sequence these events occur and which of these constitute the bottleneck in damage probing and recognition is unknown.Here, we report the first dynamical study of XPC bound to DNA containing a model (3-bp mismatch) lesion. To monitor nucleotide flipping, we positioned 2-aminopurine (2AP) within the 3-bp mismatch. To monitor more subtle motions such as DNA unwinding, we incorporated a pair of newly developed cytosine analog FRET probes, tCo (donor) and tCnitro (acceptor), on either side of the 3-bp mismatch. Upon binding of XPC to the lesion site, the 2AP fluorescence increased and the FRET efficiency between the tCo/tCnitro probes changed, as expected from the known structures of XPC-free and -bound DNA. Laser T-jump measurements on this complex revealed, for the first time, kinetics of nucleotide-flipping and DNA unwinding, with a relaxation time of 2-10 ms in the temperature range 13-39o C. Notably, a mutant XPC lacking the Beta-hairpin domain could also induce FRET change in mismatch-DNA, but could not induce an increase in 2AP fluorescence.