Pierucci O, Rickert M, Helmstetter C E
Department of Experimental Biology, Roswell Park Memorial Institute, Buffalo, New York 14263.
J Bacteriol. 1989 Jul;171(7):3760-6. doi: 10.1128/jb.171.7.3760-3766.1989.
Initiation of DNA replication from oriC in Escherichia coli takes place at a specific time in the cell division cycle, whether the origin is located on a chromosome or a minichromosome, and requires participation of the product of the dnaA gene. The effects of overproduction of DnaA protein on the cell cycle specificity of the initiation event were determined by using minichromosome replication as the assay system. DnaA protein was overproduced by inducing the expression of plasmid-encoded dnaA genes under control of either the ptac or lambda pL promoter. Induction of DnaA protein synthesis caused a burst of minichromosome replication in cells at all ages in the division cycle. The magnitude of the burst was consistent with the initiation of one round of replication per minichromosome in all cells. The replication burst was followed by a period of reduced minichromosome replication, with the reduction being greater at 30 than at 41 degrees C. The results support the idea that the DnaA protein participates in oriC replication at a stage that is limiting for initiation. Excess DnaA protein enabled all cells to achieve the state required for initiation of DNA polymerization by either effecting or overriding the normal limiting process.
在大肠杆菌中，无论oriC位于染色体还是微型染色体上，DNA复制从oriC起始都发生在细胞分裂周期的特定时间，并且需要dnaA基因产物的参与。通过使用微型染色体复制作为检测系统，确定了过量表达DnaA蛋白对起始事件细胞周期特异性的影响。通过在ptac或λ pL启动子控制下诱导质粒编码的dnaA基因表达，使DnaA蛋白过量表达。诱导DnaA蛋白合成导致在分裂周期中所有年龄段的细胞中微型染色体复制激增。激增的幅度与所有细胞中每个微型染色体一轮复制的起始一致。复制激增之后是微型染色体复制减少的时期，在30℃时减少幅度大于41℃时。结果支持这样的观点，即DnaA蛋白在起始限制阶段参与oriC复制。过量的DnaA蛋白通过影响或超越正常的限制过程，使所有细胞达到启动DNA聚合所需的状态。
