Although the mechanisms that precisely time initiation of chromosome replication in bacteria remain unclear, most clock models are based on accumulation of the active initiator protein, DnaA-ATP. During each cell division cycle, sufficient DnaA-ATP must become available to interact with a distinct set of low affinity recognition sites in the unique chromosomal replication origin, oriC, and assemble the pre-replicative complex (orisome) that unwinds origin DNA and helps load the replicative helicase. The low affinity oriC-DnaA-ATP interactions are required for the orisome's mechanical functions, and may also play a role in timing of new rounds of DNA synthesis. To further examine this possibility, we constructed chromosomal oriCs with equal preference for DnaA-ADP or DnaA-ATP at one or more low affinity recognition sites, thereby lowering the DnaA-ATP requirement for orisome assembly, and measured the effect of the mutations on cell cycle timing of DNA synthesis. Under slow growth conditions, mutation of any one of the six low affinity DnaA-ATP sites in chromosomal oriC resulted in initiation earlier in the cell cycle, but the shift was not equivalent for every recognition site. Mutation of τ2 caused a greater change in initiation age, suggesting its occupation by DnaA-ATP is a temporal bottleneck during orisome assembly. In contrast, during rapid growth, all origins with a single mutated site displayed wild-type initiation timing. Based on these observations, we propose that E. coli uses two different, DnaA-ATP-dependent initiation timing mechanisms; a slow growth timer that is directly coupled to individual site occupation, and a fast growth timer comprising DnaA-ATP and additional factors that regulate DnaA access to oriC. Analysis of origins with paired mutated sites suggests that Fis is an important component of the fast growth timing mechanism.
Keywords: DNA binding proteins; DNA replication; DnaA; cell cycle; oriC; orisomes; pre-replicative complexes; replication origin.