Oxidative damage-induced PCNA complex formation is efficient in xeroderma pigmentosum group A but reduced in Cockayne syndrome group B cells
Oxidative damage-induced PCNA complex formation is efficient in xeroderma pigmentosum group A but reduced in Cockayne syndrome group B cells
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DOI：
10.1093/nar/27.22.4476
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发表时间：
1999-11-15
影响因子：
14.9
通讯作者：
Bohr, VA
中科院分区：
文献类型：
--
作者：
Balajee, AS;Dianova, I;Bohr, VA
增殖细胞核抗原（PCNA）是DNA聚合酶δ和β的持续合成因子，对DNA复制和修复至关重要。PCNA在核苷酸切除修复（NER）的再合成步骤中是必需的。在紫外线照射后，PCNA移位成不溶性蛋白质复合物，最有可能与核基质相关。以前没有研究过在体内是否PCNA复合物的形成也发生在氧化应激后。在这项研究中，我们检测了PCNA在氧化性DNA损伤修复中的作用。PCNA复合物的形成研究了正常人细胞处理后，过氧化氢，产生各种氧化DNA损伤。PCNA的检测采用免疫荧光法和Western blot法。我们观察到，PCNA重新分配从可溶性DNA结合的形式在氧化DNA损伤的修复。分析了两种DNA修复缺陷的人天然突变细胞系：着色性干皮病A组（XP-A）和Cockayne综合征B组（CS-B）中PCNA复合物的形成。XP-A细胞在整个基因组NER中有缺陷，而CS-B细胞仅在活性基因的优先修复中有缺陷。PCNA复合物形成的免疫荧光检测在正常和XP-A细胞中相似，但在CS-B细胞中减少。与该观察结果一致，CS-B细胞中的蛋白质印迹分析显示与正常和XP-A细胞相比，PCNA重新定位的比率降低。在氧化损伤后的XP-A细胞中观察到的有效PCNA复合物形成表明，PCNA依赖性修复灶的形成可能不需要XPA基因产物。在CS-B细胞中观察到的PCNA复合物形成减少表明这些细胞在氧化DNA损伤的处理中有缺陷。
Proliferating cell nuclear antigen (PCNA), a processivity factor for DNA polymerases delta and epsilon, is essential for both DNA replication and repair. PCNA is required in the resynthesis step of nucleotide excision repair (NER). After UV irradiation, PCNA translocates into an insoluble protein complex, most likely associated with the nuclear matrix. It has not previously been investigated in vivo whether PCNA complex formation also takes place after oxidative stress. in this study, we have examined the involvement of PCNA in the repair of oxidative DNA damage. PCNA complex formation was studied in normal human cells after treatment with hydrogen peroxide, which generates a variety of oxidative DNA lesions. PCNA was detected by two assays, immunofluorescence and western blot analyses. We observed that PCNA redistributes from a soluble to a DNA-bound form during the repair of oxidative DNA damage. PCNA complex formation was analyzed in two human natural mutant cell lines defective in DNA repair: xeroderma pigmentosum group A (XP-A) and Cockayne syndrome group B (CS-B). XP-A cells are defective in overall genome NER while CS-B cells are defective only in the preferential repair of active genes. Immunofluorescent detection of PCNA complex formation was similar in normal and XP-A cells, but was reduced in CS-B cells. Consistent with this observation, western blot analysis in CS-B cells showed a reduction in the ratio of PCNA relocated as compared to normal and XP-A cells. The efficient PCNA complex formation observed in XP-A cells following oxidative damage suggests that formation of PCNA-dependent repair foci may not require the XPA gene product. The reduced PCNA complex formation observed in CS-B cells suggests that these cells are defective in the processing of oxidative DNA damage.