Kalejta R F, Hamlin J L
Department of Biochemistry, University of Virginia School of Medicine, Charlottesvile 22098, USA.
Mol Cell Biol. 1996 Sep;16(9):4915-22. doi: 10.1128/MCB.16.9.4915.
The neutral/neutral two-dimensional (2-D) gel replicon mapping technique has been used to great advantage to localize and characterize origins of replication. Interestingly, many yeast origins display a composite pattern consisting of both a bubble arc and a single-fork arc. Moreover, in every instance in which neutral/neutral 2-D gels have been used to analyze origins in higher eukaryotic cells, two or more adjacent fragments display these composite patterns. We believe that composite patterns signal inefficient origin usage in yeast cells because the replicators in question are not active in every cell cycle and in higher eukaryotic replicons because initiation sites are chosen from among many potential sites lying within a zone. However, others have suggested that the single-fork arcs in these composite gel patterns arise from nicking activity that converts replication bubbles to branched structures that comigrate with bona fide single forks. Here, we have used three different replicon mapping strategies to show that broken simian virus 40 replication bubbles trace unique arcs that are clearly distinguishable from classic, intact single forks. Thus, it is likely that composite 2-D gel patterns represent origins that are inefficiently utilized.
中性/中性二维（2-D）凝胶复制子图谱技术已被大量用于定位和表征复制起点。有趣的是，许多酵母复制起点呈现出由气泡弧和单叉弧组成的复合模式。此外，在每一个使用中性/中性二维凝胶分析高等真核细胞复制起点的实例中，两个或更多相邻片段呈现出这些复合模式。我们认为复合模式表明酵母细胞中复制起点的使用效率低下，因为所讨论的复制子并非在每个细胞周期都活跃，而在高等真核生物复制子中，起始位点是从一个区域内的许多潜在位点中选择的。然而，其他人提出这些复合凝胶模式中的单叉弧源于切口活性，这种活性将复制泡转化为与真正单叉共迁移的分支结构。在这里，我们使用了三种不同的复制子图谱策略来表明断裂的猿猴病毒40复制泡追踪独特的弧，这些弧与经典的完整单叉明显不同。因此，复合二维凝胶模式很可能代表了利用效率低下的复制起点。
