Wickner S, McKenney K
Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
J Biol Chem. 1987 Sep 25;262(27):13163-7.
Supercoiled DNA containing the replication origin of bacteriophage lambda can be replicated in vitro. This reaction requires purified lambda O and P replication proteins and a partially purified mixture of Escherichia coli proteins (Tsurimoto, T., and Matsubara, K. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7639-7643; Wold, M. S., Mallory, J.B., Roberts, J. D., LeBowitz, J. H., and McMacken, R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6176-6180). The lambda origin region has four repeats of a 19-base pair sequence to which O protein binds. To the right of these sites on the lambda map is a 40-base pair region that is rich in adenine and thymine, followed by a 28-base pair palindromic sequence. To define more precisely the boundaries of the lambda origin, we cloned a 358-base pair piece of lambda DNA containing the origin region into M13mp8 in both orientations. In vitro replication of RF I DNAs prepared from cells infected with these two M13 ori lambda phage was dependent on lambda O and P proteins and a crude protein fraction from uninfected E. coli; with these conditions there was no replication of M13mp8 RF I DNA. We made deletions from the left and the right ends of the lambda origin DNA and determined the deletion end points by DNA sequencing. We have tested RF I DNAs prepared from cells infected with phage carrying ori lambda deletions for their ability to function as templates for O- and P-dependent replication in vitro. Our results show that lambda DNA between nucleotide positions 39072 and 39160 is required for efficient O- and P-dependent replication. This 89-base pair piece of DNA includes only two of the four 19-base pair O protein-binding sites (the two right-most) and the adjoining adenine- and thymine-rich region to the right of the O-binding sites.
含有噬菌体λ复制起点的超螺旋DNA能够在体外进行复制。该反应需要纯化的λO和P复制蛋白以及大肠杆菌蛋白的部分纯化混合物（鹤本彻、松原和夫（1982年）《美国国家科学院院刊》79卷，7639 - 7643页；沃德、马洛里、罗伯茨、勒博维茨、麦克马肯（1982年）《美国国家科学院院刊》79卷，6176 - 6180页）。λ起点区域有四个19碱基对序列的重复片段，O蛋白可与之结合。在λ图谱上这些位点的右侧是一个富含腺嘌呤和胸腺嘧啶的40碱基对区域，接着是一个28碱基对的回文序列。为了更精确地界定λ起点的边界，我们将一段包含起点区域的358碱基对的λDNA片段以两种方向克隆到M13mp8中。从感染这两种M13 ori λ噬菌体的细胞中制备的RF I DNA的体外复制依赖于λO和P蛋白以及来自未感染大肠杆菌的粗蛋白组分；在这些条件下，M13mp8 RF I DNA不会复制。我们从λ起点DNA的左端和右端进行缺失，并通过DNA测序确定缺失端点。我们测试了从感染携带ori λ缺失的噬菌体的细胞中制备的RF I DNA作为O和P依赖的体外复制模板的功能能力。我们的结果表明，核苷酸位置39072和39160之间的λDNA对于高效的O和P依赖复制是必需的。这段89碱基对的DNA片段仅包含四个19碱基对O蛋白结合位点中的两个（最右侧的两个）以及O结合位点右侧相邻的富含腺嘌呤和胸腺嘧啶的区域。
