UV-Induced Ubiquitylation of XPC Protein Mediated by UV-DDB-Ubiquitin Ligase Complex

Abstract

The xeroderma pigmentosum group C (XPC) protein complex plays a key role in recognizing DNA damage throughout the genome for mammalian nucleotide excision repair (NER). Ultraviolet light (UV)-damaged DNA binding protein (UV-DDB) is another complex that appears to be involved in the recognition of NER-inducing damage, although the precise role it plays and its relationship to XPC remain to be elucidated. Here we show that XPC undergoes reversible ubiquitylation upon UV irradiation of cells and that this depends on the presence of functional UV-DDB activity. XPC and UV-DDB were demonstrated to interact physically, and both are polyubiquitylated by the recombinant UV-DDB-ubiquitin ligase complex. The polyubiquitylation altered the DNA binding properties of XPC and UV-DDB and appeared to be required for cell-free NER of UV-induced (6-4) photoproducts specifically when UV-DDB was bound to the lesion. Our results strongly suggest that ubiquitylation plays a critical role in the transfer of the UV-induced lesion from UV-DDB to XPC.

Dynamic two-stage mechanism of versatile DNA damage recognition by xeroderma pigmentosum group C protein

“…DDB1, on the other hand, is an adaptor that connects the Cul4A-RBX1 ubiquitin ligase 15 to WD40-repeat target proteins [63,64], including DDB2 itself [65,66]. Other known substrates of the Cul4A-RBX1-DDB1 ubiquitin ligase include XPC [67] as well as the core histones H2A, H3 and H4 [68,69].…”

“…Two major hypotheses have been forwarded for the mechanism by which UV-DDB contributes to the recognition of UV lesions. The handover hypothesis has been proposed on the basis of in vitro assays indicating that ubiquitin modulates the DNA-binding affinity of DDB2 and XPC [67]. In this scenario, UV-DDB recognizes UV lesions and recruits XPC protein through direct protein-protein interactions.…”

“…In this scenario, UV-DDB recognizes UV lesions and recruits XPC protein through direct protein-protein interactions. Subsequently, polyubiquitylation of DDB2 reduces its affinity for DNA and results in degradation, whereas polyubiquitylation of XPC preserves its affinity for the DNA substrate [67]. The chromatin remodeling hypothesis is prompted by the observation that DDB2 is not absolutely required for the excision of CPDs by reconstituted GGR systems using naked DNA as the substrate [74].…”

Impaired repair of cyclobutane pyrimidine dimers in human keratinocytes deficient in p53 and p63

“…For analysis of XPC and DDB2, cells were lysed and total protein was extracted and quantified as reported (38). Extracts containing equivalent amounts of total protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted and probed as described previously (8), using monoclonal antibodies against p63 (1:200, clone 4A4, Santa Cruz Biotechnology), p53 (1:500, clone DO-1, Santa Cruz Biotechnology), XPC (clone 3.26, 1:1000, GeneTex, San Antonio, TX), β-actin (clone AC-74, 1:3000, Sigma, St. Louis, MO) and a rabbit polyclonal antibody to DDB2 (1:500, gift from J.M.…”

Other Proteins Interacting with XP Proteins

“…Unlike centrin 2, however, XPE is required for efficient GG-NER activity. 11 The involvement of XPC in GG-NER is regulated also by its sumoylation with the small ubiquitin-like SUMO-1 modifier; this modification is promoted by the recruitment of XPA to the damage site. Sumoylation of XPC is necessary to prevent UV-induced degradation of XPC by the 26S proteosome system 12 as well as the dissociation of XPC from the damage site.…”