DNA repair factor XPC is modified by SUMO-1 and ubiquitin following UV irradiation
Authors: Qianzheng Zhu
Qi-En Wang
Gulzar Wani
Jinyou Li
Altaf A Wani
Mohamed A El-Mahdy
Journal: Nucleic Acids Research (2005)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Nucleotide excision repair; XPC protein; UV irradiation; DNA damage; SUMO-1
Affiliations: Department of Radiology, The Ohio State University 103 Wiseman Hall, 400 W. 12th Avenue, Columbus, OH 43210, USA.

Abstract:  Nucleotide excision repair (NER) is the major DNA repair process that removes diverse DNA lesions including UV-induced photoproducts. There are more than 20 proteins involved in NER. Among them, XPC is thought to be one of the first proteins to recognize DNA damage during global genomic repair (GGR), a sub-pathway of NER. In order to study the mechanism through which XPC participates in GGR, we investigated the possible modifications of XPC protein upon UV irradiation in mammalian cells. Western blot analysis of cell lysates from UV-irradiated normal human fibroblast, prepared by direct boiling in an SDS lysis buffer, showed several anti-XPC antibody-reactive bands with molecular weight higher than the original XPC protein. The reciprocal immunoprecipitation and siRNA transfection analysis demonstrated that XPC protein is modified by SUMO-1 and ubiquitin. By using several NER-deficient cell lines, we found that DDB2 and XPA are required for UV-induced XPC modifications. Interestingly, both the inactivation of ubiquitylation and the treatment of proteasome inhibitors quantitatively inhibited the UV-induced XPC modifications. Furthermore, XPC protein is degraded significantly following UV irradiation in XP-A cells in which sumoylation of XPC does not occur. Taken together, we conclude that XPC protein is modified by SUMO-1 and ubiquitin following UV irradiation and these modifications require the functions of DDB2 and XPA, as well as the ubiquitin-proteasome system. Our results also suggest that at least one function of UV-induced XPC sumoylation is related to the stabilization of XPC protein. 

Retracted: No

PubPeer comments:
 Figure 4A. https: imgur.com mc3T7Fs , 
 About ten months ago, I was approached about a whistleblower complaint for a figure of one of our old 2005 papers. I promptly responded to the query and received an acknowledgment about my satisfactory response erasing the concern. I thought the matter was appropriately closed. Yesterday, I was alerted by a colleague that the query was still open on the PubPeer site and my response was not added. So, I am taking this opportunity to also upload my response at this site. I am sorry for the delay as I should have personally put a parallel response here. Anyway, better late than never. Please see our response below to the concern implying Figure Splicing. First regarding the concerned Figure 4A, with blue arrows, it doesn t match the original published version. The alleged aberration is somehow amplified in the marked figure. This notwithstanding, I do see an indication of vertical lines pointed by blue arrows. Upon continuous enlargement we found that such vertical lines become similarly prominent between all the lanes. The whistle blower appears to focus only on the lines that to him her appear surreptitious. It is implied that we must have falsified the data by inserting lane 4, or entire right side lanes 4, 5 and 6 in Figure 4A. Notice that the protein bands for main XPB and nonspecific band below it match 100% and are in perfect proportion to XPC levels shown above them. Technically speaking, I would think that it would take an impossible effort to splice these so perfectly. To what end, may one ask? What could we have gained. Now scientifically speaking, artificial splicing of bands in the alleged lane here would not have provided us any advantage in terms of data presentation or conclusions. For the results of this figure, in the absence and presence of UV, we don t report any difference for 041 cell extracts or even other two cell lines within the figure. The scientific focus here is on the modified XPC bands of lanes 1 and 2, which show a clear, unambiguous and dramatic difference for the OSU-2 cells that are DDB2 competent and not in control 041 and XPE cells that lack DDB2. So, in the description of the data we only point to this very fact without harping about the lanes 3-6 results to create a phenomenon of sorts or making a major issue of the XPB levels in control cells line within the alleged lane 4. Besides, the results of the control cell line in lanes 3 and 4 are corroborated by the results of second control cell line within lanes 5 and 6. More importantly, exactly similar lane 4 results are shown in the separate Figure 4C. No so-called aberrations there. In our ongoing laboratory experiments, and published data, we must have shown the relative presence of XPC and XPB in hundreds of Westerns with identical results. So, why would we select this particular figure for any fraudulent presentation. Please take a closer look at the  C in the same Figure 4 and see for yourself the nature of XPC and XPB bands. It is nice to look at trees but looking at forest can be more meaningful. On the one hand, I really resent this unfounded finger pointing through this alert. But on the other hand I admit that there is certainly a need for scientific scrutiny and even admire the scientific whistle blowers for their efforts to keep the enterprise clean. It is our responsibility to respond to any and all concerns regarding our publications. Whereas I can understand the reason for the anonymity of the whistle blower here, I would be happy yet to erase any of his her lingering concerns directly, if needed. Feel free to reach out to me. Regards, Al Wani , 
 24 Nov 2021 correction. https: academic.oup.com nar advance-article doi 10.1093 nar gkab1165 6438029 The authors regret the presence of undisclosed image splicing in the western blots shown in Figure 4A of their article (1). Original Figure 4A. The XPB band 4 has been spliced in. The initial Western blot (XPB image shown below) included 8 samples. The last two samples (lanes 7, 8) were duplicates of 041 UV- and 041 UV+ (lanes 3, 4) in reverse order, for the purpose of Western blotting quality control. The XPB band (loading control) in 041 UV + sample (lane 4) did not blot well. Another XPB band from the duplicate loading of the same sample (lane 7) was used to replace this band. Original XPB blot u2013 In the published figure, the 041 UV + band (4th from the left) has been replaced by the duplicate lane (7th lane) New Figure 4. A. p53 and DDB2 are involved in UV-induced XPC modification by SUMO-1 and ubiquitin (A) OSU-2, p53-deficient 041 and DDB2-deficient XP-E cells were UV irradiated at 20 J m2 and incubated for 1 h. The cell lysates were subjected to western blotting with anti-XPC antibody. This change does not affect the results, discussion and conclusions presented in the article. REFERENCES 1.tWang Qi-En, Zhu Q., Wani G., El-Mahdy M.A., Li J., Wani A.A. DNA repair factor XPC is modified by SUMO-1 and ubiquitin following UV irradiation. Nucleic Acids Res. 2005; 33:4023u20134034. ,