Role of PCNA in Repair of Radiation-induced DNA Damage

如其他地方所述，这项资助的研究已经在国际期刊上发表了三篇论文。研究结果总结如下：1。用免疫荧光法对电离辐射诱导的人二倍体成纤维细胞中PCNA-DNA复合物的形成进行了表征。复合物的形成依赖于ATP和温度，而不依赖于蛋白质合成或DNA聚合酶δ / ε的活性。该复合物在辐照后12-15 h丢失，这意味着完成了依赖于pnas的DNA合成，而当蛋白质合成或DNA聚合酶δ epsilon的活性受到抑制时，它仍然存在。着色性干皮病a组患者的细胞具有核苷酸切除修复（NER）完全缺乏的特征，其时间过程与正常细胞完全相同。这些结果表明，电离辐射诱导的DNA损伤修复需要新合成的蛋白质和DNA聚合酶δ /epsilon活性来进行DNA合成，并且起始步骤与ner2不同。我们利用缺乏DNA内切酶的XP-F和XP-G细胞确定了在NER中形成PCNA复合物的步骤。这两种细胞均未显示PCNA复合物的形成，这表明该复合物是在NER过程中DNA损伤的3‘和5’侧协调调节的切口后形成的。利用XP-A细胞的不同突变，对XPA功能对PCNA复合体形成的影响进行了突变分析。C4型锌指结构域的突变完全失去了紫外线诱导的复合物形成，而c2h2型锌指结构域的突变不影响复合物的形成。这些结果与先前报道的NER能力密切相关。这表明PCNA与XPA蛋白的功能密切相关。

The study supported by this grant has aleady been published as three papers in international journals as described elsewhere. The results are summarized as follows :1. The ionizing-radiation induced PCNA-DNA complex formation was characterized in human diploid fibroblasts using an immunofluorescence method. It was revealed that complex formation is dependent on ATP and temperature, but not on protein synthesis or or activities of DNA polymerases delta/epsilon. The complex was lost 12-15 h after irradiation, which implies completion of the PCNA-dependent DNA synthesis, while it persisted when protein synthesis or activities of DNA polymerases delta epsilon were inhibited. Cells derived from a xeroderma pigmentosum group A patient, characteristic of complete deficiency in nucleotide excision repair (NER) , showed exactly the same time course as that in normal cells. These findings suggest that the repair of ionizing-radiation induced DNA damge requires newly synthesized proteins and DNA polymerases delta/epsilon activities to carry out DNA synthesis, and the initiation step is different from that in NER.2. We pinpointed at which steps the PCNA complex is formed in NER employing XP-F and XP-G cells deficient in DNA endonucleases. Neither of cells showed PCNA complex formation, implicating that the complex is formed after coordinately regulated nicks at 3'and 5'sides of the DNA lesions in the NER process.3. Mutational analysis of the XPA function on PCNA complex formation was performed using various mutations of XP-A cells. Mutations in the C4 type zinc finger domain completely lost the UV-induced complex formation, but those in the C2H2-like zinc finger domain did not affect the formation. These results were well correlated to the capacities of NER as reported previously. It was thus demonstrated that PCNA is closely linked the function of the XPA protein.