Wang Qi-en, Zhu Qianzheng, Wani Manzoor A, Wani Gulzar, Chen Jianming, Wani Altaf A
Department of Radiology, The Ohio State University, 103 Wiseman Hall, 400 W. 12th Avenue, Columbus, OH 43210, USA.
DNA Repair (Amst). 2003 May 13;2(5):483-99. doi: 10.1016/s1568-7864(03)00002-8.
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.
功能性肿瘤抑制因子p53主要在高效的全基因组修复（GGR）中发挥作用，GGR是核苷酸切除修复（NER）的一个子途径。在本研究中，在原位修复功能正常和缺陷的人类细胞中，研究了p53对XPC和TFIIH向DNA损伤位点的时空募集的调节作用。通过对完整细胞离散的亚核区域进行微孔紫外线照射诱导光产物，并通过受损DNA亚核斑点（SNS）的免疫荧光强度和密度检测特定损伤以及募集的修复因子。在照射的核灶内的SNS处原位观察到环丁烷嘧啶二聚体（CPD）和6-4光产物（6-4PP）。原位修复动力学表明，p53野生型正常成纤维细胞对CPD和6-4PP的修复均 proficient，而p53缺失的李-弗劳梅尼综合征（LFS）成纤维细胞不能有效地修复CPD，但能修复6-4PP。NER因子的共定位实验表明，在正常人细胞中，XPC和TFIIH迅速有效地募集到SNS内的DNA损伤处。相比之下，在p53缺失或p53功能受损的细胞中，XPC和TFIIH向SNS内DNA损伤处的募集效率要低得多。p53野生型和缺失型细胞中XPC和XPB的总细胞水平相似，紫外线照射后24小时内保持不变。结果还表明，募集的XPC和TFIIH从DNA损伤SNS处的分散发生在DNA损伤后的短时间内。这种分散需要功能性的XPA、XPF和XPG蛋白。综上所述，结果表明p53在GGR过程中的损伤识别和随后的修复机制组装中起显著作用，并且XPC和TFIIH向CPD的募集是p53依赖性的。这种p53作用的最可能机制是通过其下游效应蛋白DDB2。 （注：原文中“proficient”未翻译完整，可能是“ proficient in”之类的表述，这里根据上下文推测为“对……有修复能力”，翻译可能存在不准确之处，需结合完整原文进一步确认。）
