Site-directed mutagenesis for DnaA

来自新复制起点的DNA的再复制通常被抑制，使得DNA复制与细胞分裂的偶联以有序的方式进行。然而，即使在原核细胞中，抑制的分子机制仍然未知。在我以前的研究中，我证明了DnaA蛋白（ANA在大肠杆菌细胞中复制的起始因子）的内在ATP酶活性通过使DnaA功能失活而在抑制再复制中起主要作用。我还发现了DnaA ATP酶活性的刺激因子，并表明该因子仅在DNA复制开始后才被激活。基于这些结果，我提出了一个新的模型，抑制大肠杆菌细胞中的重新启动。详细地说，我们构建了ATP酶活性特异性降低的突变DnaA蛋白，并表明突变DnaA蛋白的诱导引起细胞中染色体DNA复制的过度起始，导致显性致死表型。这些观察结果表明，DnaA蛋白的内在ATP酶活性参与抑制细胞从新复制的起点重新启动

Re-replication of DNA from a newly replicated origin is normally suppressed so that coupling of DNA replication with cell division proceeds in an orderly manner. However, the molecular mechanisms for suppression remains unknown, even in prokaryotic cells. In my previous research, I demonstrated that the intrinsic ATPase activity of DnaA protein, the initiation factor for ANA replication in Escherichia coli cells, play a major role in the suppression ofre-replication by inactivating the DnaA function. I also found a stimulation factor for the DnaA ATPase activity and showed that the factor was activated only after the initiation of DNA replication. Based on these results, I proposed a new model for the suppression of re-initiation in Escherichia coli cells. In details, we constructed mutant DnaA protein whose ATPase activity is specifically decreased and showed that induction of a mutated DnaA protein caused over-initiation of chromosomal DNA replication in cells, resulting in a dominant lethal phenotype. These observations suggest that the intrinsic ATPase activity of DnaA protein is involved in the suppression of re-initiation from newly replicated origin in cells