We present evidence for a complex regulatory interplay between the initiation of DNA replication and deoxyribonucleotide synthesis. In Escherichia coli, the ATP-bound DnaA protein initiates chromosomal replication. Upon loading of the b-clamp subunit (DnaN) of the replicase, DnaA is inactivated as its intrinsic ATPase activity is stimulated by the protein Hda. The b-subunit acts as a matchmaker between Hda and DnaA. Chain elongation of DNA requires a sufficient supply of deoxyribonucleotides (dNTPs), which are produced by ribonucleotide reductase (RNR). We present evidence suggesting that the molecular switch from ATP-DnaA to ADP-DnaA is a critical step coordinating DNA replication with increased deoxyribonucleotide synthesis. Characterization of dnaA and dnaN mutations that result in a constitutively high expression of RNR reveal this mechanism. We propose that the nucleotide bound state of DnaA regulates the transcription of the genes encoding ribonucleotide reductase (nrdAB). Accordingly, the conversion of ATP-DnaA to ADP-DnaA after initiation and loading of the b-subunit DnaN would allow increased nrdAB expression, and consequently, coordinated RNR synthesis and DNA replication during the cell cycle.

SummaryIn Escherichia coli , the SeqA protein is known as a negative regulator of chromosome replication. This protein is also suggested to have a role in chromosome organization. SeqA preferentially binds to hemimethylated DNA and is by immunofluorescence microscopy seen as foci situated at the replication factories. Loss of SeqA leads to increased negative supercoiling of the DNA. We show that purified SeqA protein bound to fully methylated, covalently closed or nicked circular DNA generates positive supercoils in vitro in the presence of topoisomerase I or ligase respectively. This means that binding of SeqA changes either the twist or the writhe of the DNA. The ability to affect the topology of DNA suggests that SeqA may take part in the organization of the chromosome in vivo . The topology change performed by SeqA occurred also on unmethylated plasmids. It is, however, reasonable to suppose that in vivo the major part of such activity is performed on hemi-methylated DNA at the replication factories and presumably forms the basis for the characteristic SeqA foci observed by fluorescence microscopy.

The Escherichia coli SeqA protein binds preferentially to hemimethylated DNA and is required for inactivation (sequestration) of newly formed origins. A mutant SeqA protein, SeqA4 (A25T), which is deficient in origin sequestration in vivo, was found here to have lost the ability to form multimers, but could bind as dimers with wild-type affinity to a pair of hemimethylated GATC sites. In vitro, binding of SeqA dimers to a plasmid first generates a topology change equivalent to a few positive supercoils, then the binding leads to a topology change in the "opposite" direction, resulting in a restraint of negative supercoils. Binding of SeqA4 mutant dimers produced the former effect, but not the latter, showing that a topology change equivalent to positive supercoiling is caused by the binding of single dimers, whereas restraint of negative supercoils requires multimerization via the N-terminus. In vivo, mutant SeqA4 protein was not capable of forming foci observed by immunofluorescence microscopy, showing that N-terminus-dependent multimerization is required for building SeqA foci. Overproduction of SeqA4 led to partially restored initiation synchrony, indicating that origin sequestration may not depend on efficient higher-order multimerization into foci, but do require a high local concentration of SeqA.