Wickner S, Hoskins J, Chattoraj D, McKenney K
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1990 Jul 15;265(20):11622-7.
The mini-P1 plasmid origin of replication is contained on a 246 base pair (bp) piece of DNA. At one end there are five 19-bp binding sites for the P1 initiator protein, RepA, and near the other end there are two 9-bp DnaA protein-binding sites. To further define the limits of the origin, we cloned the origin region in M13 and constructed deletions of either end. We sequenced the DNA and tested the replicative form I DNA of the deletion phages for their ability to support RepA-dependent DNA replication in an in vitro system. The origin that is functional in vitro could be reduced to 202 bp. It includes three intact and one incomplete RepA-binding sites at one end and the two DnaA-binding sites at the other end. When the two naturally occurring DnaA-binding sites were replaced with one or two synthetic sites, only the construction containing two sites was active in vitro. We found that the minimal origin that is functional in vivo contains all of the five RepA and the two DnaA-binding sites. Mini-P1 plasmid replication both in vivo and in vitro requires two initiator proteins, the Escherichia coli DnaA protein and the P1 RepA protein. We have found that the ADP form of DnaA is as active as the ATP form of the protein in the in vitro replication of mini-P1. In contrast, only the ATP form is active for in vitro replication of plasmids carrying the E. coli origin (Bramhill, D., and Kornberg, A. (1988) Cell 52, 743-755).
微型P1质粒复制起点包含在一段246个碱基对（bp）的DNA片段上。一端有五个19bp的P1起始蛋白RepA结合位点，另一端附近有两个9bp的DnaA蛋白结合位点。为了进一步确定复制起点的界限，我们将复制起点区域克隆到M13中，并构建了两端的缺失体。我们对DNA进行测序，并测试缺失噬菌体的复制型I DNA在体外系统中支持RepA依赖性DNA复制的能力。在体外具有功能的复制起点可缩减至202bp。它一端包括三个完整和一个不完整的RepA结合位点，另一端包括两个DnaA结合位点。当两个天然存在的DnaA结合位点被一个或两个合成位点取代时，只有包含两个位点的构建体在体外具有活性。我们发现，在体内具有功能的最小复制起点包含所有五个RepA和两个DnaA结合位点。微型P1质粒在体内和体外的复制都需要两种起始蛋白，即大肠杆菌DnaA蛋白和P1 RepA蛋白。我们发现，在微型P1的体外复制中，DnaA的ADP形式与该蛋白的ATP形式一样具有活性。相比之下，只有ATP形式对携带大肠杆菌复制起点的质粒的体外复制具有活性（布拉姆希尔，D.，和科恩伯格，A.（1988年）《细胞》52卷，743 - 755页）。
