DNA damage search mechanism using single-molecule imaging

Abstract

DNA damage repair is critical for the genome maintenance. Especially, searching for DNA damage is crucial because it initiates the entire repair process. In nucleotide excision repair (NER) that is a conserved and versatile repair mechanism, XPC finds DNA lesions and recruits downstream factors. Structural studies revealed the molecular feature of damage identification by XPC, and single-molecule approach reported the diffusion of XPC along DNA. However, how XPC can recognize the defects on DNA during its diffusion and what factors affect the diffusive motion remain elusive. To elucidate the molecular mechanism underlying damage search of XPC, we directly imaged the motion of human XPC-Rad23B (hXPC-RAD23B) on undamaged or lesion-containing DNA using a high-throughput single-molecule technique, called DNA curtain. We observed the heterogeneity in motions of XPC-RAD23B, exhibiting diffusive, constrained, and immobile species. We found that the heterogeneity comes from the interaction between XPC-RAD23B and transient opening of duplex due to DNA breathing in AT-rich regions. Moreover, the diffusion coefficient dramatically increases according to ionic strength, suggesting that XPC-RAD23B diffuses along DNA via hopping, facilitating to bypass protein obstacles upon collision on DNA. We also found that XPC-RAD23B recognized cyclobutane pyrimidine dimers (CPDs) with low efficiency, proposing that another factor is necessitated. Taken together, our results give insight into how XPC-RAD23B can rapidly search for DNA damage under the crowded environments in billions of base pairs of human genome.