Ackerman E J, Iakoucheva L M
Pacific Northwest National Laboratory, Richland, Washington, 99352-0999, USA.
Methods. 2000 Oct;22(2):188-93. doi: 10.1006/meth.2000.1060.
We have developed efficient DNA repair extracts derived from the unusually large nuclei of Xenopus oocytes. These extracts use nucleotide excision repair (NER) to completely remove bulky adducts from DNA. There is very little or no synthesis on control, undamaged DNA, indicating the extracts do not have significant nonspecific nuclease activity, and repair of cyclobutane pyrimidine dimers (CPDs) occurs in the dark, indicating that NER, and not photolyase, is responsible for CPD repair. The extracts can be inactivated with antibodies specific to repair proteins and then repair activity can be restored by adding purified recombinant protein. Here we describe detailed protocols for preparing Xenopus nuclear repair extracts.
我们已经从非洲爪蟾卵母细胞异常大的细胞核中开发出了高效的DNA修复提取物。这些提取物利用核苷酸切除修复（NER）从DNA中完全去除大分子加合物。在对照的未受损DNA上几乎没有或没有合成，这表明提取物没有显著的非特异性核酸酶活性，并且环丁烷嘧啶二聚体（CPD）的修复在黑暗中发生，这表明负责CPD修复的是NER，而不是光解酶。提取物可以用针对修复蛋白的特异性抗体使其失活，然后通过添加纯化的重组蛋白来恢复修复活性。在这里，我们描述了制备非洲爪蟾核修复提取物的详细方案。
