Wu F, Goldberg I, Filutowicz M
Department of Bacteriology, University of Wisconsin, Madison 53706.
Nucleic Acids Res. 1992 Feb 25;20(4):811-7. doi: 10.1093/nar/20.4.811.
A dnaA 'null' strain could not support replication of intact plasmid R6K or derivatives containing combinations of its three replication origins (alpha, gamma, beta). DnaA binds in vitro to sites in two functionally distinct segments of the central gamma origin. The 277-bp core segment is common to all three origins and contains DnaA box 2, which cannot be deleted without preventing replication. Immediately to the left of the core lies the 106-bp origin enhancer, which contains DnaA box 1. When the origin enhancer is deleted, the core alone can still initiate replication if levels of wt pi protein are decreased or if copy-up pi mutant proteins are provided in trans. DnaA does not effect expression of R6K replication initiator protein pi, although several DnaA boxes were identified in the coding segment of the pir gene, which encodes pi. Together these data suggest that a single DnaA box, 2, is sufficient for initiation from the gamma origin and might be sufficient for initiation from the gamma origin and might be sufficient and required for the activity of the alpha and beta origins as well. Implications of the DnaA protein binding to two domains of the gamma origin and the role of the 106-bp origin enhancer in replication are discussed.
一个dnaA“缺失”菌株无法支持完整质粒R6K或含有其三个复制起点（α、γ、β）组合的衍生物的复制。DnaA在体外与中央γ起点两个功能不同的片段中的位点结合。277bp的核心片段是所有三个起点共有的，包含DnaA框2，若不阻止复制则不能删除该框。核心片段左侧紧邻106bp的起点增强子，其包含DnaA框1。当起点增强子被删除时，如果野生型π蛋白水平降低或提供反式的复制增强型π突变蛋白，仅核心片段仍可启动复制。尽管在编码π的pir基因的编码片段中鉴定出了几个DnaA框，但DnaA并不影响R6K复制起始蛋白π的表达。这些数据共同表明，单个DnaA框2足以从γ起点起始复制，可能也足以从γ起点起始复制，并且对于α和β起点的活性可能也是充分且必要的。本文讨论了DnaA蛋白与γ起点两个结构域结合的意义以及106bp起点增强子在复制中的作用。
