Slater S, Wold S, Lu M, Boye E, Skarstad K, Kleckner N
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Cell. 1995 Sep 22;82(6):927-36. doi: 10.1016/0092-8674(95)90272-4.
The seqA gene negatively modulates replication initiation at the E. coli origin, oriC. seqA is also essential for sequestration, which acts at oriC and the dnaA promoter to ensure that replication initiation occurs exactly once per chromosome per cell cycle. Initiation is promoted by full methylation of GATC sites clustered in oriC; sequestration is specific to the hemimethylated forms generated by replication. SeqA protein purification and DNA binding are described. SeqA interacts with fully methylated oriC strongly and specifically. This reaction requires multiple molecules of SeqA and determinants throughout oriC, including segments involved in open complex formation. SeqA interacts more strongly with hemimethylated DNA; in this case, oriC and non-oriC sequences are bound similarly. Also, binding of hemimethylated oriC by membrane fractions is due to SeqA. Direct interaction of SeqA protein with the replication origin is likely to be involved in both replication initiation and sequestration.
seqA基因对大肠杆菌复制起点oriC处的复制起始起负调控作用。seqA对于隔离也是必需的，隔离作用于oriC和dnaA启动子，以确保每个细胞周期每条染色体恰好发生一次复制起始。oriC中聚集的GATC位点的完全甲基化促进起始；隔离作用于复制产生的半甲基化形式。文中描述了SeqA蛋白的纯化和DNA结合。SeqA与完全甲基化的oriC强烈且特异性地相互作用。该反应需要多个SeqA分子以及oriC上的多个决定簇，包括参与开放复合物形成的片段。SeqA与半甲基化DNA的相互作用更强；在这种情况下，oriC和非oriC序列的结合方式相似。此外，膜组分对半甲基化oriC的结合是由于SeqA。SeqA蛋白与复制起点的直接相互作用可能参与复制起始和隔离过程。
