Pre-replication assembly of E. coli replisome components.
The localization of SeqA, thymidylate synthase, DnaB (helicase) and the DNA polymerase components alpha and tau, 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, alpha and tau 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 tau co-labelled in the cell centre, though not at presumed pre-replication assembly sites. By contrast, alpha and tau co-labelled consistently suggesting that they are already associated before replication starts.
den Blaauwen
Tanneke
T
Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, the Netherlands.
Aarsman
Mirjam E G
ME
Wheeler
Linda J
LJ
Nanninga
Nanne
N
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D053488:DnaB Helicases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry; D051738:Origin Recognition Complex; D013940:Thymidylate Synthase / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C114650:OriC chromosomal replication origin; D051738:Origin Recognition Complex; C089746:SeqA protein, E coli; D013940:Thymidylate Synthase; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; C506730:dnaB protein, E coli; D053488:DnaB Helicases