title: Cullin 4A-mediated proteolysis of DDB2 protein at DNA damage sites regulates in vivo lesion recognition by XPC.
abstract: Xeroderma pigmentosum (XP) complementation group E gene product, damaged DNA-binding protein 2 (DDB2), is a subunit of the DDB heterodimeric protein complex with high specificity for binding to a variety of DNA helix-distorting lesions. DDB is believed to play a role in the initial step of damage recognition in mammalian nucleotide excision repair (NER) of ultraviolet light (UV)-induced photolesions. It has been shown that DDB2 is rapidly degraded after cellular UV irradiation. However, the relevance of DDB2 degradation to its functionality in NER is still unknown. Here, we have provided evidence that Cullin 4A (CUL-4A), a key component of CUL-4A-based ubiquitin ligase, mediates DDB2 degradation at the damage sites and regulates the recruitment of XPC and the repair of cyclobutane pyrimidine dimers. We have shown that CUL-4A can be identified in a UV-responsive protein complex containing both DDB subunits. CUL-4A was visualized in localized UV-irradiated sites together with DDB2 and XPC. Degradation of DDB2 could be blocked by silencing CUL-4A using small interference RNA or by treating cells with proteasome inhibitor MG132. This blockage resulted in prolonged retention of DDB2 at the subnuclear DNA damage foci within micropore irradiated cells. Knock down of CUL-4A also decreased recruitment of the damage recognition factor, XPC, to the damaged foci and concomitantly reduced the removal of cyclobutane pyrimidine dimers from the entire genome. These results suggest that CUL-4A mediates the proteolytic degradation of DDB2 and that this degradation event, initiated at the lesion sites, regulates damage recognition by XPC during the early steps of NER.
authors.affiliation: Department of Radiology, The Ohio State University, Columbus, Ohio 43210.
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: El-Mahdy
authors.affiliation: Department of Radiology, The Ohio State University, Columbus, Ohio 43210.
authors.forename: Qianzheng
authors.initials: Q
authors.lastname: Zhu
authors.affiliation: Department of Radiology, The Ohio State University, Columbus, Ohio 43210.
authors.forename: Qi-En
authors.initials: QE
authors.lastname: Wang
authors.affiliation: Department of Radiology, The Ohio State University, Columbus, Ohio 43210.
authors.forename: Gulzar
authors.initials: G
authors.lastname: Wani
authors.affiliation: Department of Radiology, The Ohio State University, Columbus, Ohio 43210.
authors.forename: Mette
authors.initials: M
authors.lastname: Prætorius-Ibba
authors.affiliation: Department of Radiology, The Ohio State University, Columbus, Ohio 43210; Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio 43210; James Cancer Hospital and Solove Research Institute, The Ohio State University, Columbus, Ohio 43210. Electronic address: wani.2@osu.edu.
authors.forename: Altaf A
authors.initials: AA
authors.lastname: Wani
mesh_terms: D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020868:Gene Silencing; D006801:Humans; D007976:Leupeptins; D011740:Pyrimidine Dimers / Q000378:metabolism; D015398:Signal Transduction; D025801:Ubiquitin / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: C114234:CUL4A protein, human; D002843:Chromatin; D044842:Cullin Proteins; C091435:DDB1 protein, human; C117669:DDB2 protein, human; D004268:DNA-Binding Proteins; D007976:Leupeptins; D011740:Pyrimidine Dimers; D025801:Ubiquitin; C086984:XPC protein, human; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde