Bardwell L, Cooper A J, Friedberg E C
Department of Pathology, University of Texas Southwestern Medical Center, Dallas 75235.
Mol Cell Biol. 1992 Jul;12(7):3041-9. doi: 10.1128/mcb.12.7.3041-3049.1992.
The RAD1 and RAD10 genes of Saccharomyces cerevisiae are two of at least seven genes which are known to be required for damage-specific recognition and/or damage-specific incision of DNA during nucleotide excision repair. RAD1 and RAD10 are also involved in a specialized mitotic recombination pathway. We have previously reported the purification of the RAD10 protein to homogeneity (L. Bardwell, H. Burtscher, W. A. Weiss, C. M. Nicolet, and E. C. Friedberg, Biochemistry 29:3119-3126, 1990). In the present studies we show that the RAD1 protein, produced by in vitro transcription and translation of the cloned gene, specifically coimmunoprecipitates with the RAD10 protein translated in vitro or purified from yeast. Conversely, in vitro-translated RAD10 protein specifically coimmunoprecipitates with the RAD1 protein. The sites of this stable and specific interaction have been mapped to the C-terminal regions of both polypeptides. This portion of RAD10 protein is evolutionarily conserved. These results are the first biochemical evidence of a specific association between any eukaryotic proteins genetically identified as belonging to a recombination or DNA repair pathway and suggest that the RAD1 and RAD10 proteins act at the same or consecutive biochemical steps in both nucleotide excision repair and mitotic recombination.
酿酒酵母的RAD1和RAD10基因是已知的至少七个基因中的两个，这些基因在核苷酸切除修复过程中对DNA损伤特异性识别和/或损伤特异性切割是必需的。RAD1和RAD10也参与一种特殊的有丝分裂重组途径。我们之前报道过已将RAD10蛋白纯化至同质状态（L. 巴德韦尔、H. 布尔切尔、W. A. 魏斯、C. M. 尼科莱特和E. C. 弗里德伯格，《生物化学》29:3119 - 3126，1990年）。在本研究中，我们表明，通过对克隆基因进行体外转录和翻译产生的RAD1蛋白，能与体外翻译或从酵母中纯化的RAD10蛋白特异性地共免疫沉淀。相反，体外翻译的RAD10蛋白也能与RAD1蛋白特异性地共免疫沉淀。这种稳定且特异性相互作用的位点已被定位到两种多肽的C末端区域。RAD10蛋白的这一部分在进化上是保守的。这些结果是首次关于任何被基因鉴定为属于重组或DNA修复途径的真核蛋白之间存在特异性关联的生化证据，并表明RAD1和RAD10蛋白在核苷酸切除修复和有丝分裂重组中处于相同或连续的生化步骤。
