Pre-replication assembly of E. coli replisome components
Article first published online: 25 SEP 2006
Volume 62, Issue 3, pages 695–708, November 2006
How to Cite
Den Blaauwen, T., Aarsman, M. E. G., Wheeler, L. J. and Nanninga, N. (2006), Pre-replication assembly of E. coli replisome components. Molecular Microbiology, 62: 695–708. doi: 10.1111/j.1365-2958.2006.05417.x
- Issue published online: 25 SEP 2006
- Article first published online: 25 SEP 2006
- Accepted 30 August, 2006.
The localization of SeqA, thymidylate synthase, DnaB (helicase) and the DNA polymerase components α and τ, 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, α and τ 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 τ co-labelled in the cell centre, though not at presumed pre-replication assembly sites. By contrast, α and τ co-labelled consistently suggesting that they are already associated before replication starts.