Damage Search Mechanism of Human NER Protein XPC-Rad23B

Abstract

DNA damage repair is crucial for maintaining genomic integrity. Finding DNA lesions initiates the entire repair process. In nucleotide excision repair (NER), XPC recognizes DNA lesions and recruits following NER components. Structural, biochemical, and single-molecule studies revealed the molecular features of damage identification by yeast XPC ortholog Rad4. However, how XPC recognizes DNA defects have remained elusive. We directly visualized the motion of XPC-Rad23B on undamaged and lesion-containing DNA using DNA curtain, which is a high-throughput single-molecule imaging technique. We observed heterogeneity in one-dimensional motion of XPC-Rad23B along DNA, including diffusive, constrained, and immobile motion. Consecutive AT-tracks led to increase in proteins with constrained or immobile motion. The diffusion coefficient dramatically increased according to ionic strength, suggesting that XPC-Rad23B diffuse along DNA via hopping, allowing XPC-Rad23B to bypass protein obstacles during the search for DNA damage. When XPC-Rad23B identifies cyclobutane pyrimidine dimers (CPDs) during diffusion, XPC-Rad23B makes futile attempts to bind to CPDs, consistent with low CPD recognition efficiency. The low recognition efficiency and unstable binding highlight the necessity of another factor for CPD recognition. Taken together, our results provide new insight into how XPC-Rad23B can rapidly search for local defects on DNA in long human genome.