Verma R, Campbell J L
Braun Laboratories, California Institute of Technology, Pasadena 91125.
J Biol Chem. 1992 Jan 25;267(3):1648-54.
Using oligonucleotide affinity chromatography with DNase I footprinting as an assay we have looked for proteins that interact with sequence elements within the yeast origin of replication, autonomously replicating sequence 1 (ARS1). In this work we describe a protein that binds with high affinity to DNA but displays only moderate sequence specificity. It is eluted at 0.7 M salt from an ARS1 oligonucleotide column. Footprinting analysis on ARS1 at a high protein concentration revealed at least three sites of protection flanking element A and its repeats. Element A itself is rendered hypersensitive to DNase I digestion upon protein binding. This pattern is also observed for the H4 and HMR-E ARSs, suggesting that the protein alters the DNA conformation at element A and its repeats. The affinity-purified fraction is also capable of supercoiling a relaxed, covalently closed plasmid in the presence of topoisomerase. Highly purified preparations of the protein are enriched in an 18-kDa polypeptide which can be renatured from a denaturing gel and shown to bind ARS1 DNA. We have designated this protein DBF-A, DNA-binding factor A.
我们使用以DNase I足迹法为检测手段的寡核苷酸亲和色谱法，寻找与酵母复制起点自主复制序列1（ARS1）内的序列元件相互作用的蛋白质。在这项工作中，我们描述了一种与DNA具有高亲和力但序列特异性仅为中等的蛋白质。它在0.7M盐浓度下从ARS1寡核苷酸柱上洗脱下来。在高蛋白浓度下对ARS1进行足迹分析，发现在元件A及其重复序列两侧至少有三个保护位点。蛋白质结合后，元件A本身对DNase I消化变得高度敏感。在H4和HMR - E ARS中也观察到这种模式，这表明该蛋白质改变了元件A及其重复序列处的DNA构象。亲和纯化的组分在拓扑异构酶存在的情况下，也能够使松弛的、共价闭合的质粒超螺旋化。该蛋白质的高度纯化制剂富含一种18 kDa的多肽，该多肽可从变性凝胶中复性，并显示能结合ARS1 DNA。我们将这种蛋白质命名为DBF - A，即DNA结合因子A。
