Guzder Sami N, Sommers Christopher H, Prakash Louise, Prakash Satya
Sealy Center for Molecular Science, University of Texas Medical Branch at Galveston, 6.104 Blocker Medical Research Building, 11th and Mechanic Streets, Galveston, TX 77555-1061, USA.
Mol Cell Biol. 2006 Feb;26(3):1135-41. doi: 10.1128/MCB.26.3.1135-1141.2006.
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 rad1Delta 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.
真核生物中的核苷酸切除修复（NER）需要大量蛋白质因子在损伤位点组装，然后这些因子协同对受损DNA链进行双切口切割。然而，不同蛋白质因子在损伤位点的组装方式仍不清楚。此前，我们已经表明，在酿酒酵母中，NER蛋白作为不同蛋白质亚组装体的组成部分存在：例如，Rad1-Rad10核酸酶与损伤识别蛋白Rad14形成紧密复合物，并且Rad1-Rad10-Rad14复合物可以从酵母细胞中完整纯化出来。由于Rad1-Rad10核酸酶对结合DNA中的紫外线损伤没有特异性，与Rad14的结合可能为将Rad1-Rad10核酸酶靶向体内损伤位点提供一种有效手段。为了验证这一想法的有效性，我们在此鉴定了两个rad1突变，这些突变使酵母细胞对紫外线的敏感性与rad1Delta突变相同，但对Rad1的重组功能没有影响。通过对这些rad1突变进行的遗传和生化研究，我们得出结论，Rad1-Rad10核酸酶与Rad14形成复合物的能力对于将该核酸酶靶向体内损伤位点至关重要。我们讨论了这些观察结果对于不同NER蛋白在损伤位点组装方式的意义。
