Guzder S N, Sung P, Prakash L, Prakash S
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1061, USA.
J Biol Chem. 1997 Aug 29;272(35):21665-8. doi: 10.1074/jbc.272.35.21665.
In eukaryotes, nucleotide excision repair of ultraviolet light-damaged DNA is a highly intricate process that requires a large number of evolutionarily conserved protein factors. Genetic studies in the yeast Saccharomyces cerevisiae have indicated a specific role of the RAD7 and RAD16 genes in the repair of transcriptionally inactive DNA. Here we show that the RAD7- and RAD16-encoded products exist as a complex of 1:1 stoichiometry, exhibiting an apparent dissociation constant (Kd) of <4 x 10(-10) M. The Rad7-Rad16 complex has been purified to near homogeneity in this study and is shown to bind, in an ATP-dependent manner and with high specificity, to DNA damaged by ultraviolet light. Importantly, inclusion of the Rad7-Rad16 complex in the in vitro nucleotide excision repair system that consists entirely of purified components results in a marked stimulation of damage specific incision. Thus, Rad7-Rad16 complex is the ATP-dependent DNA damage sensor that specifically functions with the ensemble of nucleotide excision repair factor (NEF) 1, NEF2, NEF3, and replication protein A in the repair of transcriptionally inactive DNA. We name this novel complex of Rad7 and Rad16 proteins NEF4.
在真核生物中，紫外线损伤DNA的核苷酸切除修复是一个高度复杂的过程，需要大量进化上保守的蛋白质因子。对酿酒酵母的遗传学研究表明，RAD7和RAD16基因在转录非活性DNA的修复中具有特定作用。在此我们表明，RAD7和RAD16编码的产物以化学计量比1:1的复合物形式存在，其表观解离常数（Kd）<4×10^(-10) M。在本研究中，Rad7-Rad16复合物已被纯化至接近均一性，并显示以ATP依赖的方式且具有高特异性地结合紫外线损伤的DNA。重要的是，将Rad7-Rad16复合物加入完全由纯化成分组成的体外核苷酸切除修复系统中，会显著刺激损伤特异性切口。因此，Rad7-Rad16复合物是ATP依赖的DNA损伤传感器，在转录非活性DNA的修复中与核苷酸切除修复因子（NEF）1、NEF2、NEF3和复制蛋白A协同发挥特定功能。我们将Rad7和Rad16蛋白的这种新型复合物命名为NEF4。
