title: Pre-replication assembly of E. coli replisome components.
abstract: 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.
authors.affiliation: Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, the Netherlands.
authors.forename: Tanneke
authors.initials: T
authors.lastname: den Blaauwen
authors.forename: Mirjam E G
authors.initials: ME
authors.lastname: Aarsman
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Wheeler
authors.forename: Nanne
authors.initials: N
authors.lastname: Nanninga
mesh_terms: 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
chemical_list: 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