Jansen L E, Verhage R A, Brouwer J
MGC Department of Molecular Genetics, Leiden Institute of Chemistry, Leiden University, P. O. Box 9502, 2300 RA Leiden, The Netherlands.
J Biol Chem. 1998 Dec 11;273(50):33111-4. doi: 10.1074/jbc.273.50.33111.
The yeast Rad4 and Rad23 proteins form a complex that is involved in nucleotide excision repair (NER). Their function in this process is not known yet, but genetic data suggest that they act in an early step in NER. We have purified an epitope-tagged Rad4.Rad23 (tRad4. Rad23) complex from yeast cells, using a clone overproducing Rad4 with a hemagglutinin-tag at its C terminus. tRad4.Rad23 complex purified by both conventional and immuno-affinity chromatography complements the in vitro repair defect of rad4 and rad23 mutant extracts, demonstrating that these proteins are functional in NER. Using electrophoretic mobility shift assays, we show preferential binding of the tRad4.Rad23 complex to damaged DNA in vitro. UV-irradiated, as well as N-acetoxy-2-(acetylamino)fluorene-treated DNA, is efficiently bound by the protein complex. These data suggest that Rad4.Rad23 interacts with DNA damage during NER and may play a role in recognition of the damage.
酵母Rad4和Rad23蛋白形成一个参与核苷酸切除修复（NER）的复合物。它们在此过程中的功能尚不清楚，但遗传数据表明它们在NER的早期步骤中发挥作用。我们使用一个在其C末端带有血凝素标签的过量表达Rad4的克隆，从酵母细胞中纯化了一个带有表位标签的Rad4.Rad23（tRad4.Rad23）复合物。通过常规和免疫亲和层析纯化的tRad4.Rad23复合物弥补了rad4和rad23突变体提取物的体外修复缺陷，表明这些蛋白在NER中具有功能。使用电泳迁移率变动分析，我们在体外显示了tRad4.Rad23复合物与受损DNA的优先结合。紫外线照射以及经N-乙酰氧基-2-（乙酰氨基）芴处理的DNA能被该蛋白复合物有效结合。这些数据表明Rad4.Rad23在NER过程中与DNA损伤相互作用，并且可能在损伤识别中发挥作用。
