Purification of PCNA as a nucleotide excision repair protein.
Purification of PCNA as a nucleotide excision repair protein.
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DOI：
10.1093/nar/20.10.2441
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发表时间：
1992-05
影响因子：
14.9
通讯作者：
A. Nichols;A. Sancar
中科院分区：
文献类型：
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作者：
A. Nichols;A. Sancar
人细胞游离提取物在体外进行核苷酸切除修复。萃取物很容易在DEAE色谱柱上分离成两部分。低盐馏分（0.1 M KCl）和高盐馏分（0.8 M KCl）均不具有修复合成能力，但两者结合可恢复修复合成活性。通过修复合成实验，我们从高盐部分中纯化了一个37 kDa的蛋白，该蛋白与低盐部分一起恢复了修复合成活性。氨基酸序列分析、氨基酸组成和PCNA抗体免疫印迹显示，37 kDa蛋白是已知的刺激DNA聚合酶delta和epsilon的增殖细胞核抗原（PCNA）。通过使用一种专门测量胸腺嘧啶二聚体切除的实验，我们发现PCNA本身并不需要实际的切除反应，但通过使切除修复酶催化地翻转来增加切除的程度。
Human cell free extracts carry out nucleotide excision repair in vitro. The extract is readily separated into two fractions by chromatography on a DEAE column. Neither the low salt (0.1 M KCl) nor the high salt (0.8 M KCl) fractions are capable of repair synthesis but the combination of the two restore the repair synthesis activity. Using the repair synthesis assay we purified a protein of 37 kDa from the high salt fraction which upon addition to the low salt fraction restores repair synthesis activity. Amino acid sequence analysis, amino acid composition and immunoblotting with PCNA antibodies revealed that the 37 kDa protein is the proliferating cell nuclear antigen (PCNA) known to stimulate DNA Polymerases delta and epsilon. By using an assay which specifically measures the excision of thymine dimers we found that PCNA is not required for the actual excision reaction per se but increases the extent of excision by enabling the excision repair enzyme to turn over catalytically.