Karmakar P, Balajee A S, Natarajan A T
MGC, Department of Radiation Genetics and Chemical Mutagenesis, Leiden University, PO Box 9503, 2300 RA Leiden, Wassenaarseweg 72, The Netherlands.
Mutagenesis. 2001 May;16(3):225-32. doi: 10.1093/mutage/16.3.225.
Proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA polymerase delta and epsilon, is involved in both DNA replication and repair. Previous studies in vitro have demonstrated the requirement of PCNA in the resynthesis step of nucleotide excision repair (NER) and base excision repair (BER). Using a native chromatin template isolated under near physiological conditions, we have analysed the involvement of PCNA in the BER pathway in different NER defective human cell lines. The repair sites and PCNA were visualized by indirect immunolabelling followed by fluorescence microscopy. The results indicate that exposure to X-rays triggers the induction of PCNA in all the three human fibroblast cell lines studied, namely normal, xeroderma pigmentosum group A (XP-A) and Cockayne syndrome group B (CS-B). In all the cell lines, induction of PCNA and repair patches occurred in a dose- and time-dependent fashion. Induction of repair patches in NER-deficient XP-A cells suggests that the X-ray-induced lesions are largely repaired via the BER pathway involving PCNA as one of the key components of this pathway. X-ray-induced repair synthesis was greatly inhibited by treatment of cells with DNA polymerase inhibitors aphidicolin and cytosine arabinoside. Interestingly, inhibition of repair resynthesis did not affect the intensity of PCNA staining in X-irradiated cells indicating that the PCNA may be required for the BER pathway at a step preceding the resynthesis step.
增殖细胞核抗原（PCNA）是DNA聚合酶δ和ε的辅助因子，参与DNA复制和修复过程。以往的体外研究表明，PCNA在核苷酸切除修复（NER）和碱基切除修复（BER）的再合成步骤中是必需的。我们使用在接近生理条件下分离的天然染色质模板，分析了PCNA在不同NER缺陷型人类细胞系的BER途径中的作用。通过间接免疫标记和荧光显微镜观察修复位点和PCNA。结果表明，暴露于X射线会在所有研究的三种人类成纤维细胞系中诱导PCNA的表达，这三种细胞系分别是正常细胞系、A型着色性干皮病（XP - A）细胞系和B型科凯恩综合征（CS - B）细胞系。在所有细胞系中，PCNA的诱导和修复斑块的形成呈剂量和时间依赖性。在NER缺陷的XP - A细胞中诱导修复斑块表明，X射线诱导的损伤主要通过涉及PCNA作为该途径关键成分之一的BER途径进行修复。用DNA聚合酶抑制剂阿非迪霉素和阿糖胞苷处理细胞，可大大抑制X射线诱导的修复合成。有趣的是，修复再合成的抑制并不影响X射线照射细胞中PCNA染色的强度，这表明PCNA可能在再合成步骤之前的BER途径步骤中是必需的。
