Molecular Mechanism for Initiation of DNA Replication of ColE2 and Co1E3 Plasmids

ColE2和ColE3的基本复制子由三个功能部分组成：一个针对质粒特异性启动子蛋白的基因(Rep；33-35kda)；一个能够在Rep蛋白存在的情况下启动复制的起始点(32和33bp)；一个针对抗原RNA的基因(RNA1；Rep蛋白与起始点相互作用的特异性是由每个质粒的Rep蛋白羧基末端附近的20个氨基酸区域和起始端的单个碱基对缺失(ColE2)或插入(ColE3)决定的。部分纯化的ColE2 Rep蛋白通过过滤器结合的方法被证明特异性地结合到ColE2的起始处。复制从包含克隆起点的区域开始，并单向进行。新合成的前导链的5‘末端定位在起始点内的固定位置。新合成的滞后链的3‘端定位于起始点的下游端。这似乎是单向复制的基本机制。新合成的前导链在其5‘端含有3个核苷酸残基(PpApGpA)，ColE2 Rep蛋白与宿主DNA聚合酶I和单链DNA结合蛋白一起在相同的位置与相同的引物RNA特异性地启动了前导链的合成。该反应除了需要ATP和GTP外，还特别需要ADP，并将ADP整合到引物RNA中。Rep蛋白似乎是一种新的序列特异性启动子酶，能够在双链DNA上发挥作用。

The basic replicons of ColE2 and ColE3 consist of three functional portions : A gene for the plasmid-specific initiator protein (Rep ; 33-35 Kda) ; an origin (32 and 33 bp) capable of initiating replication in the presence of the Rep protein ; a gene for an antigens RNA (RNA I ; 115 n) complementary to the 5' untranslated region of the Rep mRNA and negatively regulating initiation of replication.Specificity of interaction of the Rep proteins and the origins is determined by the 20 amino acid-region near the carboxy terminal portion of the Rep protein of each plasmid and a single base pair deletion (ColE2) or insertion (ColE3) in the origin.The partially purified ColE2 Rep protein was shown to bind specifically to the ColE2 origin by using the filter-binding method.In vitro replication of ColE2 and ColE3 DNA in crude E. Coli cell extracts requires the Rep proteins and host DNA polymerase I but not RNA polymerase and DnaG primase. Replication starts from a region containing the cloned origin and proceeds unidirectionally. The 5' end of the newly synthesized leading strand was localized at a fixed position within the origin. The 3' end of the newly synthesized lagging strand was localized at the downstream end of the origin. This seems to be the basic mechanism for the unidirectional replication. The newly synthesized leading strand contains three ribonucleotide residues (ppApGpA) at its 5' end.The ColE2 Rep protein in conjunction with the host DNA polymerase I and single-strand DNA binding protein specifically starts the leading strand synthesis at the identical position with the identical primer RNA. The reaction specifically requires ADP in addition to ATP and GTP and incorporates ADP into the primer RNA. The Rep protein seems to be a novel sequence-specific primase capable of functioning on double-strand DNA.