Riber Leise, Fujimitsu Kazuyuki, Katayama Tsutomu, Løbner-Olesen Anders
Department of Science, Systems and Models, Roskilde University, DK-4000 Roskilde, Denmark.
Mol Microbiol. 2009 Jan;71(1):107-22. doi: 10.1111/j.1365-2958.2008.06516.x. Epub 2008 Nov 6.
Initiation of chromosome replication in Escherichia coli is limited by the initiator protein DnaA associated with ATP. Within the replication origin, binding sites for DnaA associated with ATP or ADP (R boxes) and the DnaA(ATP) specific sites (I-boxes, tau-boxes and 6-mer sites) are found. We analysed chromosome replication of cells carrying mutations in conserved regions of oriC. Cells carrying mutations in DnaA-boxes I2, I3, R2, R3 and R5 as well as FIS and IHF binding sites resembled wild-type cells with respect to origin concentration. Initiation of replication in these mutants occurred in synchrony or with slight asynchrony only. Furthermore, lack of Hda stimulated initiation in all these mutants. The DnaA(ATP) containing complex that leads to initiation can therefore be formed in the absence of several of the origin DnaA binding sites including both DnaA(ATP) specific I-boxes. However, competition between I-box mutant and wild-type origins, revealed a positive role of I-boxes on initiation. On the other hand, mutations affecting DnaA-box R4 were found to be compromised for initiation and could not be augmented by an increase in cellular DnaA(ATP)/DnaA(ADP) ratio. Compared with the sites tested here, R4 therefore seems to contribute to initiation most critically.
大肠杆菌中染色体复制的起始受到与ATP结合的起始蛋白DnaA的限制。在复制起点内，可发现与ATP或ADP结合的DnaA的结合位点（R盒）以及DnaA（ATP）特异性位点（I盒、tau盒和6聚体位点）。我们分析了oriC保守区域发生突变的细胞的染色体复制情况。在DnaA盒I2、I3、R2、R3和R5以及FIS和IHF结合位点发生突变的细胞，其起始浓度与野生型细胞相似。这些突变体中的复制起始仅同步发生或略有异步。此外，缺乏Hda会刺激所有这些突变体的起始。因此，在缺少包括DnaA（ATP）特异性I盒在内的几个起始DnaA结合位点的情况下，仍可形成导致起始的含DnaA（ATP）的复合物。然而，I盒突变体与野生型起点之间的竞争揭示了I盒在起始过程中的积极作用。另一方面，发现影响DnaA盒R4的突变在起始方面存在缺陷，并且细胞中DnaA（ATP）/DnaA（ADP）比率的增加无法增强其起始能力。因此，与这里测试的位点相比，R4似乎对起始的贡献最为关键。
