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Abstract
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.
Highlights
All cells must initiate new rounds of chromosomal DNA synthesis with temporal precision and only once per cell cycle to avoid genome instability
Mutation of a low affinity DnaA-ATP recognition site to allow interaction with DnaA-ADP should decrease the amount of DnaA-ATP required for orisome formation, and, if the mutation is carried in chromosomal oriC, it should shift initiation timing to earlier in the cell cycle
When DnaA-ADP is used in the reaction, only the mutant origin is fully occupied by 100 nM DnaA-ADP (Figure 1B); in contrast, binding of 100 nM DnaA-ADP binding to I3, C2, C3, I2 I1, and τ2 in wild type oriC gives a dimethyl sulfate (DMS) modification pattern similar to that seen with 40 nM DnaA-ATP (McGarry et al, 2004)
Summary
All cells must initiate new rounds of chromosomal DNA synthesis with temporal precision and only once per cell cycle to avoid genome instability. Bacteria accomplish this by using a triggering mechanism that requires the AAA+ (ATPases Associated with various cellular Activities) initiator protein, DnaA. In E. coli oriC, there are 11 DnaA binding sites within the 190 base pairs that are adjacent to the DNA Unwinding Element (DUE) (Figure 1A; Bramhill and Kornberg, 1988; Kowalski and Eddy, 1989; Rozgaja et al, 2011). Three of these sites (R1, R2, and R4), with consensus sequence, 5 -TTA/TTNCACA-3 , bind
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