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Role of PCNA in Repair of Radiation-induced DNA Damage

PCNA 在修复辐射诱导的 DNA 损伤中的作用

基本信息

批准号：
07457446
负责人：
SASAKI Takehito
金额：
$ 4.42万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

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.

这笔资助支持的研究已经在国际期刊上发表了三篇论文，如其他地方所述。研究结果如下： 1．使用免疫荧光方法对人二倍体成纤维细胞中电离辐射诱导的 PCNA-DNA 复合物形成进行了表征。结果表明，复合物的形成取决于 ATP 和温度，但不取决于蛋白质合成或 DNA 聚合酶 delta/epsilon 的活性。该复合物在辐射后12-15小时丢失，这意味着PCNA依赖性DNA合成完成，而当蛋白质合成或DNA聚合酶δε的活性受到抑制时，该复合物仍然存在。源自着色性干皮病 A 组患者的细胞，具有核苷酸切除修复（NER）完全缺陷的特征，表现出与正常细胞完全相同的时间进程。这些发现表明，电离辐射引起的DNA损伤的修复需要新合成的蛋白质和DNA聚合酶δ/ε活性来进行DNA合成，且起始步骤与NER.2中的不同。我们使用缺乏 DNA 核酸内切酶的 XP-F 和 XP-G 细胞，确定了 NER 中 PCNA 复合物形成的步骤。两种细胞均未显示PCNA复合物形成，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。3．使用 XP-A 细胞的各种突变对 XPA 功能对 PCNA 复合物形成的影响进行突变分析。 C4型锌指结构域中的突变完全丧失了紫外线诱导的复合物形成，但C2H2类锌指结构域中的突变并不影响形成。这些结果与之前报道的 NER 能力密切相关。由此证明PCNA与XPA蛋白的功能密切相关。