Pre‐replication assembly of E. coli replisome components

Abstract

SummaryThe localization of SeqA, thymidylate synthase, DnaB (helicase) and the DNA polymerase components a and t, has been studied by immunofluorescence microscopy. The origin has been labelled through GFP-LacI bound near oriC. SeqA was located in the cell centre for one replication factory (RF) and at 1/4 and 3/4 positions in pre-divisional cells harbouring two RFs. The transition of central to 1/4 and 3/4 positions of SeqA appeared abrupt. Labelled thymidylate synthetase was found all over the cell, thus not supporting the notion of a dNTP-synthesizing complex exclusively localized near the RF. More DnaB, a and t foci were found than expected. We have hypothesized that extra foci arise at pre-replication assembly sites, where the number of sites equals the number of origins, i.e. the number of future RFs. A reasonable agreement was found between predicted and found foci. In the case of multifork replication the number of foci appeared consistent with the assumption that three RFs are grouped into a higher-order structure. The RF is probably separate from the foci containing SeqA and the hemi-methylated SeqA binding sites because these foci did not coincide significantly with DnaB as marker of the RF. Co-labelling of DnaB and oriC revealed limited colocalization, indicating that DnaB did not yet become associated with oriC at a pre-replication assembly site. DnaB and t co-labelled in the cell centre, though not at presumed prereplication assembly sites. By contrast, a and t co-labelled consistently suggesting that they are already associated before replication starts.

Correlation between ribonucleoside-diphosphate reductase and three replication proteins in Escherichia coli

“…The number of DnaB helicase and DnaX τ subunit foci, also greater than that of SeqA foci, could be explained by pre-replication assembly of the replisome proteins [31]. Likewise, the greater number of NrdB foci relative to those of SeqA can be explained by assuming that this protein was also pre-assembled together with other replisome proteins before replication initiated.…”

“…On the one hand, the distributions of lengths at birth and at division are known to have coefficients of variation of 13.5 per cent [28], and consequently so do the cell sizes at any given cell age. And on the other, replication proteins can be assembled into a pre-replication structure that would lead to more foci than replicating forks [31]. Both effects could explain the number of NrdB foci found in the period between termination and initiation shown in Figure 3 where no replication forks are expected.…”

DNA and origin region segregation are not affected by the transition from rod to sphere after inhibition of Escherichia coli MreB by A22

“…Control cells that were not treated with A22 nor with mecillinam were expected to contain either two or four origins based on the DNA replication cycle of LMC500 cells (see Fig. 2B in Den Blaauwen et al ., 2006). Due to the transition from rod to sphere, the cells became shorter and fatter (Figs 3 and 4).…”

Escherichia coli Chromosomal Loci Segregate from Midcell with Universal Dynamics

“…coli possess a single, circular chromosome that is condensed into a nucleic acid and protein complex called the nucleoid (1). Chromosome replication is initiated near midcell at the origin of replication (oriC) and proceeds bidirectionally around the chromosome (2,3). Before replicated sister loci can segregate, a brief ''cohesion'' period occurs, with intercatenation linkages holding the sister chromosomes together (4)(5)(6).…”