Role of PCNA in Repair of Radiation-induced DNA Damage

由该基金支持的研究已经在其他地方描述的国际期刊上发表了三篇论文。主要研究结果如下：1.用免疫荧光法研究了电离辐射诱导人二倍体成纤维细胞PCNA-DNA复合物的形成。结果表明，复合物的形成依赖于ATP和温度，但不依赖于蛋白质的合成或DNA聚合酶δ/δ的活性。复合物在照射后12-15 h丢失，这意味着完成了依赖于PCNA的DNA合成，而当蛋白质合成或DNA聚合酶δ-κ B的活性被抑制时，复合物仍然存在。来源于着色性干皮病A组患者的细胞，其特征是核苷酸切除修复（NER）完全缺乏，表现出与正常细胞完全相同的时间过程。这些结果表明，电离辐射诱导的DNA损伤的修复需要新合成的蛋白质和DNA聚合酶δ/β活性来进行DNA合成，其起始步骤与NER 1不同.我们查明了PCNA复合物在NER中形成的步骤，采用DNA内切酶缺陷的XP-F和XP-G细胞。两种细胞均未出现PCNA复合物的形成，提示该复合物是在NER过程中DNA损伤的3 '和5'端协同调控后形成的.使用XP-A细胞的各种突变进行XPA对PCNA复合物形成的功能的突变分析。C4型锌指结构域的突变完全丧失了UV诱导的复合物形成，但C2 H2样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能力相关。结果表明，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.