Ellwood K B, Yen Y M, Johnson R C, Carey M
Department of Biological Chemistry, University of California at Los Angeles School of Medicine, Los Angeles, California 90095-1737, USA.
Mol Cell Biol. 2000 Jun;20(12):4359-70. doi: 10.1128/MCB.20.12.4359-4370.2000.
Assembly of enhanceosomes requires architectural proteins to facilitate the DNA conformational changes accompanying cooperative binding of activators to a regulatory sequence. The architectural protein HMG-1 has been proposed to bind DNA in a sequence-independent manner, yet, paradoxically, it facilitates specific DNA binding reactions in vitro. To investigate the mechanism of specificity we explored the effect of HMG-1 on binding of the Epstein-Barr virus activator ZEBRA to a natural responsive promoter in vitro. DNase I footprinting, mutagenesis, and electrophoretic mobility shift assay reveal that HMG-1 binds cooperatively with ZEBRA to a specific DNA sequence between two adjacent ZEBRA recognition sites. This binding requires a strict alignment between two adjacent ZEBRA sites and both HMG boxes of HMG-1. Our study provides the first demonstration of sequence-dependent binding by a nonspecific HMG-box protein. We hypothesize how a ubiquitous, nonspecific architectural protein can function in a specific context through the use of rudimentary sequence recognition coupled with cooperativity. The observation that an abundant architectural protein can bind DNA cooperatively and specifically has implications towards understanding HMG-1's role in mediating DNA transactions in a variety of enzymological systems.
增强体的组装需要结构蛋白来促进激活因子与调控序列协同结合时伴随的DNA构象变化。结构蛋白HMG-1被认为以序列非依赖的方式结合DNA，但矛盾的是，它在体外促进特异性DNA结合反应。为了研究特异性机制，我们在体外探索了HMG-1对爱泼斯坦-巴尔病毒激活因子ZEBRA与天然响应启动子结合的影响。DNA酶I足迹法、诱变和电泳迁移率变动分析表明，HMG-1与ZEBRA协同结合到两个相邻ZEBRA识别位点之间的特定DNA序列上。这种结合需要两个相邻ZEBRA位点与HMG-1的两个HMG框之间严格对齐。我们的研究首次证明了非特异性HMG框蛋白的序列依赖性结合。我们推测一种普遍存在的非特异性结构蛋白如何通过使用基本的序列识别和协同作用在特定环境中发挥作用。大量结构蛋白能协同且特异性结合DNA这一观察结果，对于理解HMG-1在多种酶学系统中介导DNA事务的作用具有重要意义。
