Abstract
The initiation of chromosome replication is tightly regulated in bacteria to ensure that it takes place only once per cell cycle. In many proteobacteria, this process requires the ATP-bound form of the DnaA protein. The regulatory inactivation of DnaA (RIDA) facilitates the conversion of DnaA-ATP into replication-inactive DnaA-ADP, thereby preventing overinitiation. Homologues of the HdaA protein, together with the β-clamp of the DNA polymerase (DnaN), are required for this process. Here, we used fluorescence resonance energy transfer experiments to demonstrate that HdaA interacts with DnaN in live Caulobacter crescentus cells. We show that a QFKLPL motif in the N-terminal region of HdaA is required for this interaction and that this motif is also needed to recruit HdaA to the subcellular location occupied by the replisome during DNA replication. An HdaA mutant protein that cannot colocalize or interact with DnaN can also not support the essential function of HdaA. These results suggest that the recruitment of HdaA to the replisome is needed during RIDA in C. crescentus, probably as a means to sense whether chromosome replication has initiated before DnaA becomes inactivated. In addition, we show that a conserved R145 residue located in the AAA+ domain of HdaA is also needed for the function of HdaA, although it does not affect the interaction of HdaA with DnaN in vivo. The AAA+ domain of HdaA may therefore be required during RIDA after the initial recruitment of HdaA to the replisome by DnaN.
Highlights
Proper regulation of the initiation of chromosome replication is crucial to ensure that the genome is replicated only once per cell cycle, so that cells maintain a constant number of chromosomes
An HdaA mutant protein that cannot colocalize or interact with DnaN can not support the essential function of HdaA. These results suggest that the recruitment of HdaA to the replisome is needed during regulatory inactivation of DnaA (RIDA) in C. crescentus, probably as a means to sense whether chromosome replication has initiated before DnaA becomes inactivated
As the E. coli RIDA system needs a DNA-loaded b-sliding clamp to be active in vitro (Katayama et al, 1998; Su’etsugu et al, 2004), the b-sliding clamp may be the component of the RIDA system that senses if replication is ongoing, to ensure that DnaA is inactivated only once DNA replication has started
Summary
Proper regulation of the initiation of chromosome replication is crucial to ensure that the genome is replicated only once per cell cycle, so that cells maintain a constant number of chromosomes. Each chromosome has a single origin of replication and its replication requires the initiator protein DnaA (Messer, 2002; Mott & Berger, 2007). In the gammaproteobacterium Escherichia coli, DnaA or the origin of replication (oriC) is the target of three main systems regulating the initiation of chromosomal replication (Katayama et al, 2010; Leonard & Grimwade, 2011): the titration and inactivation of DnaA molecules by the chromosomal datA locus RIDA requires the activity of two proteins in E. coli: Hda and the DNA-loaded b-sliding clamp of the DNA polymerase (DnaN)
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