Damage search mechanism of human NER protein XPC-RAD23B at the single-molecule level

Abstract

Nucleotide excision repair (NER) is the most versatile DNA repair machinery to eliminate UV-induced photo-lesions and chemical adducts on bases. Defects of NER cause serious diseases such as Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. NER is performed through multiple steps by many NER proteins and their cooperative interactions. In the multiple steps, the damage search is the most important because it is a rate limiting step to initiate the entire NER process. In human NER, XPC-RAD23B is in charge of the damage search. To investigate the damage search mechanism of XPC-RAD23B, we examined the movement of XPC-RAD23B in real time using a high-throughput single-molecule imaging technique called DNA curtain. XPC-RAD23B bound to random sequences and then moved along DNA through 1D diffusion. Interestingly, XPC-RAD23B exhibited three distinct types of motion of XPC-RAD23B: diffusive, immobile, and constrained. We revealed that the immobile state and constrained motion result from transient trapping at DNA breathings in AT-rich areas of DNA. In addition, the increase of diffusion coefficients with ionic strength suggested that XPC-RAD23B diffuses via hopping, which facilitates to search for DNA lesions rapidly by bypassing protein roadblocks on DNA. Furthermore, we examined how XPC-RAD23B identifies cyclobutane pyrimidine dimers (CPDs) during 1D diffusion and estimated the CPD recognition efficiency. Our results will give insight into the damage search mechanism of NER through crowded environments of genomic DNA.