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The SeqA protein was identified as a factor that prevents reinitiation of newly replicated, hemimethylated origins. SeqA also seems to inhibit initiation of fully methylated origins, thus contributing to the regulation of chromosomal replication. The SeqA protein was found to bind to two sites in the left part of the origin, near the AT-rich region where strand separation takes place during initiation of replication. The same binding sites seemed to be preferred irrespective of whether the origin was in the newly replicated (hemimethylated) state or not. In addition to binding specifically to groups of GATC sites, the SeqA protein was capable of interacting non-specifically with negatively supercoiled DNA, restraining the supercoils in a fashion similar to that seen with histone-like protein HU. The restraint of supercoils by SeqA was, in contrast to that of HU, cooperative.

In vivo studies suggest that the Escherichia coli SeqA protein modulates replication initiation in two ways: by delaying initiation and by sequestering newly replicated origins from undergoing re-replication. As a first approach towards understanding the biochemical bases for these effects, we have examined the effects of purified SeqA protein on replication reactions performed in vitro on an oriC plasmid. Our results demonstrate that SeqA directly affects the biochemical events occurring at oriC. First, SeqA inhibits formation of the pre-priming complex. Secondly, SeqA can inhibit replication from an established pre-priming complex, without disrupting the complex. Thirdly, SeqA alters the dependence of the replication system on DnaA protein concentration, stimulating replication at low concentrations of DnaA. Our data suggest that SeqA participates in the assembly of initiation-competent complexes at oriC and, at a later stage, influences the behaviour of these complexes.

The seqA gene negatively modulates replication initiation at the E. coli origin, oriC. seqA is also essential for sequestration, which acts at oriC and the dnaA promoter to ensure that replication initiation occurs exactly once per chromosome per cell cycle. Initiation is promoted by full methylation of GATC sites clustered in oriC; sequestration is specific to the hemimethylated forms generated by replication. SeqA protein purification and DNA binding are described. SeqA interacts with fully methylated oriC strongly and specifically. This reaction requires multiple molecules of SeqA and determinants throughout oriC, including segments involved in open complex formation. SeqA interacts more strongly with hemimethylated DNA; in this case, oriC and non-oriC sequences are bound similarly. Also, binding of hemimethylated oriC by membrane fractions is due to SeqA. Direct interaction of SeqA protein with the replication origin is likely to be involved in both replication initiation and sequestration.

The DnaC protein is required for loading the DnaB helicase at oriC. Thus DnaC promotes the formation of the pre-replication complex, but must leave the complex in order for the DnaB protein to function as a helicase. In vitro, a slight excess of DnaC inhibits the movement of replication forks by inhibiting DnaB helicase activity (Allen and Kornberg, 1991). Here we show that inhibition of DNA replication by excess DnaC also occurs in vivo. The rate of replication-fork movement was measured by flow cytometry. Initiation of replication was inhibited with rifampicin and the rate of fork movement monitored during replication runout by measuring the increase in the fraction of the cell population with fully replicated chromosomes. The replication rate was inversely related to the amount of excess DnaC protein. Initiation of replication was also inhibited. Co-overexpression of DnaB protein alleviated the inhibition of replication caused by moderate excess of DnaC. The results show that DnaC interacts with replication forks during elongation in vivo, probably by binding to DnaB and inhibiting its helicase activity. Therefore, the ratio of DnaC to DnaB and the affinity of DnaC for a helicase hexamer at an established replication fork are of great importance for the rate of replication fork movement also in vivo.

With few exceptions, the signal peptides from proteins inserted into, or translocated through, the membranes of gram-negative bacteria or the endoplasmic reticulum of eukaryotes have no sequence homologies. Therefore these signal peptides have not been considered to contain information related to the different final localizations of the proteins. In this study, 43 signal peptide amino acid sequences from proteins with different final localizations in Escherichia coli have been subjected to a multivariate data analysis. Each amino acid residue was characterized by 20 physico-chemical properties, yielding a multivariate property profile for each peptide. The similarities/dissimilarities in the property profiles for the signal peptides from different classes were compared with each other by generating few-dimensional partial least squares (PLS) discriminant plots. With this approach, signal peptides from proteins localized to the periplasmic space (PS), the outer membrane (OM), and the extracellular surroundings (excreted proteins), were separated into distinct groups. Signal peptides from pili proteins were not separated from the OM signal peptides and only partly from the PS signal peptides, but were clearly different from the signal peptides of the excreted proteins. Signal peptides from inner membrane proteins were similar to those of the PS peptides. The size and the hydrophobicity of different peptide segments were responsible for the separation of the signal peptide classes. For example, the hydrophobicity of the N-terminal segment of the signal peptides increased with an increased distance from the cytoplasm of the final localization for the corresponding proteins. Thus, many signal peptides from proteins with different final localizations in E. coli have different discernible physico-chemical profiles.