The genome is constantly confronted with the risk of DNA damage by various factors such as hazardous metabolic byproducts, carcinogens, and UV radiation. Nucleotide excision repair (NER) is one of DNA repair systems that organisms have evolved to protect the genome. Dysfunction of the NER pathway causes rare genetic disorders; xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. In mammalian global genomic NER, XPC (xeroderma pigmentosum group C complementing protein) detects DNA lesions and then recruits a ten-subunit complex, TFIIH to the lesion to open up the damaged DNA. Although TFIIH is mostly engaged in transcription as a general transcription factor under normal culture conditions, it rapidly switches to sites of DNA damage upon UV irradiation of cells. However, the precise mechanism underlying the recruitment of TFIIH to DNA lesions by XPC has remained unclear. In the present study, Okuda et al. have determined the structure of the acidic region of human XPC bound to the pleckstrin homology (PH) domain of TFIIH p62 using nuclear magnetic resonance (NMR) spectroscopy. XPC uses a coupled folding and binding mode, and wraps around the basic surface of the p62 PH domain. The bound structure of XPC closely resembles the extended acidic string-like structures observed for the general transcription factor TFIIEα and the tumor suppressor p53 bound to the p62 PH domain; however, the structure reveals critical differences in the recognition site. The key residues of XPC necessary for strong binding have been verified by mutational analyses using isothermal titration calorimetry (ITC) in vitro and immunoprecipitation in vivo. Alanine substitution of these key residues compromised the recruitment of TFIIH to sites of DNA damage, UV resistance, and the repair of UV-induced pyrimidine-pyrimidone (6-4) photoproducts in XPC-deficient cells stably transformed to express XPC protein. This study sheds light on the mechanism for functional cooperation between XPC and TFIIH in the early stage of global genomic NER.
Figure Legend: Solution structure of the complex formed between the XPC acidic string and the TFIIH p62 PH domain. Overlay of the backbone structures of the 20 best structures (ribbon representation). XPC is shown in rainbow color and TFIIH p62 in light blue.
Okuda M1, Kinoshita M2, Kakumu E2, Sugasawa K2, Nishimura Y3.[expand title=”Show Affiliations”]
- Graduate School of Medical Life Science, Yokohama City University, 1-7-29, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan
- Biosignal Research Center, Organization of Advanced Science and Technology, 1-1, Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan; Department of Biology, Graduate, School of Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan.
- Graduate School of Medical Life Science, Yokohama City University, 1-7-29, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan; [email protected][/expand]
In global genome repair (GGR), XPC detects damaged nucleotides and recruits TFIIH complex. The small acidic region of XPC binds to the pleckstrin homology (PH) domain of TFIIH subunit p62; however, the recognition mechanism remains elusive. Here, we use nuclear magnetic resonance to present the tertiary structure of XPC bound to the PH domain. The XPC acidic region forms a long string stabilized by insertion of Trp133 and Val136 into two separate hollows of the PH domain, coupled with extensive electrostatic contacts. Analysis of several XPC mutants revealed that particularly Trp133 is essential for binding to the PH domain. In cell lines stably expressing mutant XPC, alanine substitution at Trp133 or Trp133/Val136 compromised UV resistance, recruitment of TFIIH to DNA damage, and removal of UV-induced photoproducts from genomic DNA. These findings show how TFIIH complex is recruited by XPC to damaged DNA, advancing our understanding of the early stage of GGR.
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