Francesconi S C, Eisenberg S
Department of Microbiology, School of Medicine, University of Connecticut Health Center, Farmington 06032.
Mol Cell Biol. 1989 Jul;9(7):2906-13. doi: 10.1128/mcb.9.7.2906-2913.1989.
We previously identified a protein activity from Saccharomyces cerevisiae, OBF1, that bound specifically to a DNA element present in autonomously replicating sequences ARS120 and ARS121 (S. Eisenberg C. Civalier, and B. K. Tye, Proc. Natl. Acad. Sci. USA 85:743-746, 1988). OBF1 has now been purified to near homogeneity by conventional protein and DNA affinity chromatography. Electrophoresis of the purified protein in sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two polypeptides. The major protein band had a relative molecular size of 123 kilodaltons, and the minor protein band, which constituted only a small fraction of total protein, had a molecular size of 127 kilodaltons. Both polypeptides cochromatographed with the specific ARS120 DNA-binding activity and formed a stable protein-DNA complex, isolatable by sedimentation through sucrose gradients. Using antibodies, we have shown that both polypeptides are associated with the isolated protein-DNA complexes. The ARS DNA-binding activity had a Stokes radius of 54 A (5.4 nm) and a sedimentation coefficient of 4.28S, as determined by gel filtration and sedimentation through glycerol gradients, respectively. These physical parameters, together with the denatured molecular size values, suggested that the proteins exist in solution as asymmetric monomers. Since both polypeptides recognized identical sequences and had similar physical properties, they are probably related. In addition to binding to ARS120, we found that purified OBF1 bounds with equal affinity to ARS121 and with 5- and 10-fold-lower affinity to ARS1 and HMRE, respectively. Furthermore, in the accompanying paper (S. S. Walker, S. C. Francesconi, B. K. Tye, and S. Eisenberg, Mol. Cell. Biol. 9:2914-2921, 1989), we demonstrate the existence of a high, direct correlation between the ability of purify OBF1 to bind to ARS121 and optimal in vivo ARS121 activity as an origin of replication. These findings, taken together, suggest a role for OBF1 in ARS function, presumably at the level of initiation of DNA replication at the ARS.
我们之前从酿酒酵母中鉴定出一种蛋白质活性物质，即OBF1，它能特异性结合自主复制序列ARS120和ARS121中存在的一种DNA元件（S. 艾森伯格、C. 西瓦利尔和B. K. 泰伊，《美国国家科学院院刊》85:743 - 746，1988年）。现在，通过传统的蛋白质和DNA亲和色谱法，OBF1已被纯化至近乎同质。在十二烷基硫酸钠 - 聚丙烯酰胺凝胶中对纯化后的蛋白质进行电泳，结果显示存在两种多肽。主要的蛋白带相对分子质量为123千道尔顿，次要的蛋白带仅占总蛋白的一小部分，其分子大小为127千道尔顿。这两种多肽都与特异性的ARS120 DNA结合活性一起进行共色谱分离，并形成了一种稳定的蛋白质 - DNA复合物，可通过蔗糖梯度沉降法分离出来。利用抗体，我们已证明这两种多肽都与分离出的蛋白质 - DNA复合物相关。通过凝胶过滤和甘油梯度沉降法分别测定，ARS DNA结合活性的斯托克斯半径为54埃（5.4纳米），沉降系数为4.28S。这些物理参数，连同变性后的分子大小值，表明这些蛋白质在溶液中以不对称单体形式存在。由于这两种多肽识别相同的序列且具有相似的物理性质，它们可能具有相关性。除了与ARS120结合外，我们还发现纯化后的OBF1与ARS121具有同等亲和力，而与ARS1和HMRE的亲和力分别低5倍和10倍。此外，在随附的论文中（S. S. 沃克、S. C. 弗朗切斯科尼、B. K. 泰伊和S. 艾森伯格，《分子与细胞生物学》9:2914 - 2921，1989年），我们证明了纯化后的OBF1与ARS121结合的能力与ARS121作为复制起点的最佳体内活性之间存在高度直接的相关性。综合这些发现，表明OBF1在ARS功能中发挥作用，大概是在ARS处DNA复制起始的水平。
