Torheim N K, Skarstad K
Department of Cell Biology, Institute for Cancer Research, Montebello, 0310 Oslo, Norway.
EMBO J. 1999 Sep 1;18(17):4882-8. doi: 10.1093/emboj/18.17.4882.
Chromosome replication in Escherichia coli is initiated by the DnaA protein. Binding of DnaA to the origin, oriC, followed by formation of an open complex are the first steps in the initiation process. Based on in vivo studies the SeqA protein has been suggested to function negatively in the initiation of replication, possibly by inhibiting open complex formation. In vitro studies have shown that SeqA inhibits oriC-dependent replication. Here we show by KMnO(4) probing that SeqA inhibits open complex formation. The inhibition was not caused by prevention of DnaA binding to the oriC plasmids, indicating that SeqA prevented strand separation in oriC either directly, by interacting with the AT-rich region, or indirectly, by changing the topology of the oriC plasmids. SeqA was found to restrain the negative supercoils of the oriC plasmid. In comparison with the effect of HU on plasmid topology, SeqA seemed to act more cooperatively. It is likely that the inhibition of open complex formation is caused by the effect of SeqA on the topology of the plasmids. SeqA also restrained the negative supercoils of unmethylated oriC plasmids, which do not bind SeqA specifically, suggesting that the effect on topology is not dependent on binding of SeqA to a specific sequence in oriC.
大肠杆菌中的染色体复制由DnaA蛋白启动。DnaA与复制起点oriC结合，随后形成开放复合物是启动过程的第一步。基于体内研究，有人提出SeqA蛋白在复制起始过程中起负向作用，可能是通过抑制开放复合物的形成。体外研究表明SeqA抑制oriC依赖性复制。在此我们通过高锰酸钾探测表明SeqA抑制开放复合物的形成。这种抑制不是由于阻止DnaA与oriC质粒结合所致，这表明SeqA要么通过与富含AT的区域相互作用直接阻止oriC中的链分离，要么通过改变oriC质粒的拓扑结构间接阻止链分离。发现SeqA抑制oriC质粒的负超螺旋。与HU对质粒拓扑结构的影响相比，SeqA似乎起更协同的作用。开放复合物形成的抑制可能是由SeqA对质粒拓扑结构的影响引起的。SeqA还抑制未甲基化的oriC质粒的负超螺旋，这些质粒不特异性结合SeqA，这表明对拓扑结构的影响不依赖于SeqA与oriC中特定序列的结合。
