Fossum Solveig, Søreide Sølvi, Skarstad Kirsten
Department of Cell Biology, Institute for Cancer Research, Montebello, 0310 Oslo, Norway.
Mol Microbiol. 2003 Feb;47(3):619-32. doi: 10.1046/j.1365-2958.2003.t01-1-03329.x.
In Escherichia coli wild-type cells newly formed origins cannot be reinitiated. The prevention of reinitiation is termed sequestration and is dependent on the hemimethylated state of newly replicated DNA. Several mutants discovered in a screen for the inability to sequester hemimethylated origins have been mapped to the seqA gene. Here, one of these mutants, seqA2, harbouring a single amino acid change in the C-terminal end of the SeqA protein, was found to also be unable to form foci in vivo. The SeqA foci seen in the wild-type cells are believed to arise from multimerization of SeqA on hemimethylated DNA at the replication fork, presumably representing organization of newly formed DNA by SeqA. The result suggests that the process of origin sequestration is closely tied to the process of focus maintenance at the replication fork. In vitro, purified SeqA2 protein was found incapable of forming highly ordered multimers that bind hemimethylated oriC. The mutant protein was also incapable of restraining negative supercoils. Both in vivo and in vitro results support the idea that origin sequestration is an integral part of organization of newly formed DNA performed by SeqA.
在大肠杆菌野生型细胞中，新形成的复制起点不能重新起始。防止重新起始被称为隔离，并且依赖于新复制DNA的半甲基化状态。在筛选不能隔离半甲基化起点的过程中发现的几个突变体已被定位到seqA基因。在这里，这些突变体之一seqA2，在SeqA蛋白的C末端有一个单氨基酸变化，被发现也不能在体内形成焦点。野生型细胞中看到的SeqA焦点被认为是由于SeqA在复制叉处的半甲基化DNA上多聚化产生的，大概代表了SeqA对新形成DNA的组织。结果表明，起点隔离过程与复制叉处焦点维持过程密切相关。在体外，发现纯化的SeqA2蛋白不能形成结合半甲基化oriC的高度有序的多聚体。突变蛋白也不能抑制负超螺旋。体内和体外结果都支持这样的观点，即起点隔离是SeqA对新形成DNA进行组织的一个组成部分。
