Tumor suppressor p53 dependent recruitment of nucleotide excision repair factors XPC and TFIIH to DNA damage.
Functional tumor suppressor p53 is mainly required for efficient global genomic repair (GGR), a subpathway of nucleotide excisions repair (NER). In this study, the regulatory effect of p53, on the spaciotemporal recruitment of XPC and TFIIH to DNA damage sites, was investigated in repair-proficient and -deficient human cells in situ. Photoproducts were induced through micropore UV irradiation of discrete subnuclear areas of intact cells and the specific lesions, as well as recruited repair factors, were detected by immunofluorescent intensity and density of the damaged DNA subnuclear spots (SNS). Both cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP) were visualized in situ at SNS within irradiated nuclear foci. The in situ repair kinetics revealed that p53-WT normal fibroblasts are proficient for the repair of both CPD and 6-4PP, whereas, p53-Null Li-Fraumeni syndrome (LFS) fibroblasts fail to efficiently repair CPD but not 6-4PP. Colocalization experiments of the NER factors showed that in normal human cells, XPC and TFIIH are rapidly and efficiently recruited to DNA damage within SNS. By contrast, recruitment of both XPC and TFIIH to DNA damage in SNS occurred much less efficiently in p53-Null or p53-compromised cells. The total cellular levels of XPC and XPB were similar in both p53-WT and -Null cells and remained unchanged up to 24h following UV irradiation. The results also showed that dispersal of recruited XPC and TFIIH from DNA damage SNS occurs within a short period after DNA damage. Such dispersal requires functional XPA, XPF and XPG proteins. Taken together, the results demonstrated that p53 plays a pronounced role in the damage recognition and subsequent assembly of repair machinery during GGR and the recruitment of XPC and TFIIH to CPD is p53-dependent. Most likely mechanism of this p53 action is through its downstream effector protein, DDB2.
Wang
Qi-en
QE
Department of Radiology, The Ohio State University, 103 Wiseman Hall, 400 W. 12th Avenue, Columbus, OH 43210, USA.
Zhu
Qianzheng
Q
Wani
Manzoor A
MA
Wani
Gulzar
G
Chen
Jianming
J
Wani
Altaf A
AA
D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence; D013997:Time Factors; D051758:Transcription Factor TFIIH; D019473:Transcription Factors, TFII / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D014466:Ultraviolet Rays
D002843:Chromatin; D004268:DNA-Binding Proteins; D019473:Transcription Factors, TFII; D016159:Tumor Suppressor Protein p53; D051758:Transcription Factor TFIIH; C086984:XPC protein, human; D004247:DNA