Complex formation with damage recognition protein Rad14 is essential for Saccharomyces cerevisiae Rad1-Rad10 nuclease to perform its function in nucleotide excision repair in vivo
Complex formation with damage recognition protein Rad14 is essential for Saccharomyces cerevisiae Rad1-Rad10 nuclease to perform its function in nucleotide excision repair in vivo
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DOI：
10.1128/mcb.26.3.1135-1141.2006
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发表时间：
2006-02-01
影响因子：
5.3
通讯作者：
Prakash, S
中科院分区：
文献类型：
--
作者：
Guzder, SN;Sommers, CH;Prakash, S
真核生物中的核苷酸切除修复（NER）需要在损伤位点处组装大量蛋白质因子，然后这些蛋白质因子协调受损DNA链的双切口。然而，不同蛋白因子在病变部位组装的方式仍不清楚。以前，我们已经表明，在酵母酿酒酵母中，NER蛋白作为不同的蛋白酶的组分存在：例如，Rad 1-Rad 10核酸酶与损伤识别蛋白Rad 14形成紧密的复合物，并且Rad 1-Rad 10-Rad 14的复合物可以从酵母细胞中完整地纯化。由于Rad 1-Rad 10核酸酶对结合DNA中的UV损伤没有特异性，因此与Rad 14的结合可以为Rad 1-Rad 10核酸酶靶向体内损伤位点提供有效手段。为了测试这个想法的有效性，在这里，我们确定了两个rad 1突变，使酵母细胞的紫外线敏感的rad 1三角洲突变，但没有影响的重组功能的Rad 1。从我们的遗传和生物化学研究与这些rad 1突变，我们得出结论，Rad 1-Rad 10核酸酶的能力，在一个复杂的关联Rad 14是至关重要的，这种核酸酶的靶向病变部位在体内。我们讨论了这些意见的影响，通过不同的NER蛋白组装在病变部位的手段。
Nucleotide excision repair (NER) in eukaryotes requires the assembly of a large number of protein factors at the lesion site which then coordinate the dual incision of the damaged DNA strand. However, the manner by which the different protein factors are assembled at the lesion site has remained unclear. Previously, we have shown that in the yeast Saccharomyces cerevisiae, NER proteins exist as components of different protein subassemblies: the Rad1-Rad10 nuclease, for example, forms a tight complex with the damage recognition protein Rad14, and the complex of Rad1-Rad10-Rad14 can be purified intact from yeast cells. As the Rad1-Rad10 nuclease shows no specificity for binding UV lesions in DNA, association with Rad14 could provide an effective means for the targeting of Rad1-Rad10 nuclease to damage sites in vivo. To test the validity of this idea, here we identify two rad1 mutations that render yeast cells as UV sensitive as the rad1 Delta mutation but which have no effect on the recombination function of Rad1. From our genetic and biochemical studies with these rad1 mutations, we conclude that the ability of Rad1-Rad10 nuclease to associate in a complex with Rad14 is paramount for the targeting of this nuclease to lesion sites in vivo. We discuss the implications of these observations for the means by which the different NER proteins are assembled at the lesion site.