Savio M, Stivala L A, Scovassi A I, Bianchi L, Prosperi E
Istituto di Patologia Generale, Università di Pavia, Italy.
Oncogene. 1996 Oct 17;13(8):1591-8.
Evidence is presented that after exposure of normal human fibroblasts to UV-C light, nuclear binding of the proliferating cell nuclear antigen (PCNA) required for nucleotide excision repair, was rapidly triggered in the G1 and G2 phases of the cell cycle. Association to repair sites of the detergent-insoluble form of PCNA reached a peak 15-30 min after irradiation, and then decreased to basal levels within 24-48 h. In contrast, the nuclear association of p21 protein showed a slower kinetics, reaching maximal levels between 24 and 48 h but, similarly to PCNA, occurring only in G1 and G2 phases. Although the two proteins are known to be associated as detergent-soluble proteins, it is unknown whether they associate also in the detergent-insoluble form. To address this question, the chromatin-bound form of PCNA was released by using DNAse I. DNA digestion resulted in the almost complete release of PCNA from its binding sites, while only about 60% of nuclear-bound p21 could be solubilized. Immunoprecipitation of PCNA and p21 released by enzymatic digestion showed that p21 was associated with PCNA bound to late DNA repair sites. These results indicate that during nucleotide excision repair, nuclear binding of PCNA precedes that of p21 protein, and suggest that temporal association of p21 with the detergent-insoluble fraction is coincident with the disassembly of PCNA from DNA repair sites.
有证据表明，正常人成纤维细胞暴露于UV-C光后，核苷酸切除修复所需的增殖细胞核抗原（PCNA）的核结合在细胞周期的G1和G2期迅速被触发。PCNA去污剂不溶性形式与修复位点的结合在照射后15 - 30分钟达到峰值，然后在24 - 48小时内降至基础水平。相比之下，p21蛋白的核结合动力学较慢，在24至48小时之间达到最高水平，但与PCNA类似，仅在G1和G2期出现。虽然已知这两种蛋白以去污剂可溶性蛋白的形式相互关联，但它们是否也以去污剂不溶性形式相互关联尚不清楚。为了解决这个问题，使用DNA酶I释放与染色质结合的PCNA形式。DNA消化导致PCNA几乎完全从其结合位点释放，而只有约60%的核结合p21可被溶解。对酶消化释放的PCNA和p21进行免疫沉淀显示，p21与结合到晚期DNA修复位点的PCNA相关联。这些结果表明，在核苷酸切除修复过程中，PCNA的核结合先于p21蛋白的核结合，并表明p21与去污剂不溶性部分的时间关联与PCNA从DNA修复位点的解离同时发生。
