Basic Research Laboratory, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892-4260, USA.
Nucleic Acids Res. 2020 Nov 4;48(19):11016-11029. doi: 10.1093/nar/gkaa826.
Studies of bacterial chromosomes and plasmids indicate that their replication initiator proteins bind to origins of replication at many double-stranded sites and also at AT-rich regions where single-stranded DNA is exposed during origin opening. Single-strand binding apparently promotes origin opening by stabilizing an open structure, but how the initiator participates in this process and the contributions of the several binding sites remain unclear. Here, we show that the initiator protein of Vibrio cholerae specific to chromosome 2 (Chr2) also has single-strand binding activity in the AT-rich region of its origin. Binding is strand specific, depends on repeats of the sequence 5'ATCA and is greatly stabilized in vitro by specific double-stranded sites of the origin. The stability derives from the formation of ternary complexes of the initiator with the single- and double-stranded sites. An IHF site lies between these two kinds of sites in the Chr2 origin and an IHF-induced looping out of the intervening DNA mediates their interaction. Simultaneous binding to two kinds of sites in the origin appears to be a common mechanism by which bacterial replication initiators stabilize an open origin.
对细菌染色体和质粒的研究表明，它们的复制起始蛋白结合到许多双链复制起始点，也结合到富含 AT 的区域，在该区域，单链 DNA 在起始点打开时暴露。单链结合显然通过稳定开放结构来促进起始点打开，但起始蛋白如何参与该过程以及几个结合位点的贡献仍不清楚。在这里，我们表明霍乱弧菌染色体 2（Chr2）特异性的起始蛋白在其起始点的富含 AT 区域也具有单链结合活性。结合具有链特异性，依赖于序列 5'ATCA 的重复，并且在体外由起始点的特定双链位点极大地稳定。稳定性源自起始蛋白与单链和双链位点的三元复合物的形成。在 Chr2 起始点中，一个 IHF 位点位于这两种位点之间，并且 IHF 诱导的中间 DNA 环出介导它们的相互作用。在起始点同时结合两种类型的位点似乎是细菌复制起始蛋白稳定开放起始点的一种常见机制。
