Pre-replication assembly of E. coli replisome components

Authors

  • Tanneke Den Blaauwen,

    1. Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, the Netherlands.
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  • Mirjam E. G. Aarsman,

    1. Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, the Netherlands.
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  • Linda J. Wheeler,

    1. Department of Biochemistry and Biophysics, 2011 Agricultural and Life Sciences Building, Oregon State University, Corvallis, OR 97331-7305, USA.
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  • Nanne Nanninga

    Corresponding author
    1. Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, the Netherlands.
      *E-mail nanninga@science.uva.nl; Tel. (+31) 20 5255194/5187; Fax (+31) 20 5257934.
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*E-mail nanninga@science.uva.nl; Tel. (+31) 20 5255194/5187; Fax (+31) 20 5257934.

Summary

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.

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