Abstract
DNA repair is critical for maintaining genomic integrity. Finding DNA lesions initiates the entire repair process. In human nucleotide excision repair (NER), XPC-RAD23B recognizes DNA lesions and recruits downstream factors. Although previous studies revealed the molecular features of damage identification by the yeast orthologs Rad4-Rad23, the dynamic mechanisms by which human XPC-RAD23B recognizes DNA defects have remained elusive. Here, we directly visualized the motion of XPC-RAD23B on undamaged and lesion-containing DNA using high-throughput single-molecule imaging. We observed three types of one-dimensional motion of XPC-RAD23B along DNA: diffusive, immobile and constrained. We found that consecutive AT-tracks led to increase in proteins with constrained motion. The diffusion coefficient dramatically increased according to ionic strength, suggesting that XPC-RAD23B diffuses along DNA via hopping, allowing XPC-RAD23B to bypass protein obstacles during the search for DNA damage. We also examined how XPC-RAD23B identifies cyclobutane pyrimidine dimers (CPDs) during diffusion. XPC-RAD23B makes futile attempts to bind to CPDs, consistent with low CPD recognition efficiency. Moreover, XPC-RAD23B binds CPDs in biphasic states, stable for lesion recognition and transient for lesion interrogation. Taken together, our results provide new insight into how XPC-RAD23B searches for DNA lesions in billions of base pairs in human genome.
Highlights
Nucleotide excision repair (NER) is a highly conserved DNA repair pathway in charge of eliminating a diverse repertoire of DNA damage such as ultraviolet (UV) lightinduced photo-lesions, intrastrand crosslinks and bulky adducts derived from various carcinogens [1,2]
In global genome NER (GG-NER), xeroderma pigmentosum complementation group C protein (XPC) along with RAD23B and Centrin2 recognizes a variety of NER substrates by sensing the local distortion and/or thermodynamic destabilization of the DNA helix caused by the modified bases [4,5]
Full length XPC-RAD23B tagged with a 3×FLAG peptide at the N-terminus of XPC was purified and labeled with a FLAG-antibody-conjugated quantum dot (Qdot) (Figure 1A and Supplementary Figure S2A)
Summary
Nucleotide excision repair (NER) is a highly conserved DNA repair pathway in charge of eliminating a diverse repertoire of DNA damage such as ultraviolet (UV) lightinduced photo-lesions, intrastrand crosslinks and bulky adducts derived from various carcinogens [1,2]. About thirty different proteins involved in NER remove DNA lesions in an orchestrated manner [1]. In TC-NER, an RNA polymerase stalled at a lesion during transcription serves as a DNA damage indicator. In GG-NER, xeroderma pigmentosum complementation group C protein (XPC) along with RAD23B and Centrin recognizes a variety of NER substrates by sensing the local distortion and/or thermodynamic destabilization of the DNA helix caused by the modified bases [4,5]. XPC recruits TFIIH, which verifies the chemical modification of the NER substrate and opens a bubble around the lesion site using the activity of its two helicase subunits XPB and XPD. The NER process is completed by filling in the gap by DNA polymerases and sealing the nick by DNA ligases [1]
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