Jazwinski S M, Edelman G M
Proc Natl Acad Sci U S A. 1982 Jun;79(11):3428-32. doi: 10.1073/pnas.79.11.3428.
In a search for a replication complex, the activity that replicates the 2-micrometers yeast DNA plasmid in vitro was isolated in a high molecular weight form (Mr approximately 2 X 10(6) by gel filtration and rate-zonal sedimentation from extracts prepared from cells of the budding yeast Saccharomyces. When obtained from cells in late logarithmic cultures this material or "complex" was labile compared to that from early logarithmic cultures, and it did not survive as a complex after ammonium sulfate precipitation. This suggests that, as cultures approach stationary phase and cells cease growth, the association of its protein constituents may be altered. A chimera of 2-micrometers DNA inserted into the plasmid pBR322 was used to test for binding of components of the complex. After a brief incubation of the chimera in vitro with the high molecular weight material containing replicating activity, a protein "knob" was found associated with the 2-micrometers DNA as shown by electron microscopy. This association was not random but was limited to two positions on the plasmid. In the same series of experiments, the in vitro origins of 2-micrometers plasmid replication were also mapped. Two origins were found, consistent in position with those that have been identified in vivo. Molecules utilizing both origins simultaneously in vitro were not observed, and replication in vitro was bidirectional. The location of the origins corresponded to the positions at which the protein knobs associated with 2-micrometers DNA. This and the fact that no replicative intermediates with associated complexes were detected raises the possibility that a specific protein complex may be involved in initiation of DNA replication.
为寻找复制复合物，我们从出芽酵母酿酒酵母的细胞提取物中，通过凝胶过滤和速率区带沉降法，以高分子量形式（通过凝胶过滤和速率区带沉降法测得分子量约为2×10⁶）分离出了在体外复制2微米酵母DNA质粒的活性物质。当从对数生长期后期的细胞中获得这种物质或“复合物”时，与对数生长期早期细胞来源的相比，它不稳定，并且在硫酸铵沉淀后不能作为复合物存活。这表明，随着培养物接近稳定期且细胞停止生长，其蛋白质成分的结合可能会发生改变。将插入质粒pBR322的2微米DNA嵌合体用于测试复合物成分的结合情况。将嵌合体与含有复制活性的高分子量物质在体外短暂孵育后，通过电子显微镜观察发现一个蛋白质“瘤”与2微米DNA相关联。这种关联不是随机的，而是局限于质粒上的两个位置。在同一系列实验中，还对2微米质粒复制的体外起始位点进行了定位。发现了两个起始位点，其位置与在体内鉴定出的一致。未观察到在体外同时利用两个起始位点的分子，并且体外复制是双向的。起始位点的位置与与2微米DNA相关联的蛋白质瘤的位置相对应。这以及未检测到带有相关复合物的复制中间体这一事实，增加了一种特定蛋白质复合物可能参与DNA复制起始的可能性。
