Expression of the seqA gene is negatively modulated by the HU protein in Escherichia coli
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O R I GI N A L P A P E R
H. Lee á H. K. Kim á S. Kang á C. B. Hong J. Yim á D. S. Hwang
Expression of the seqA gene is negatively modulated by the HU protein in Escherichia coli Received: 23 June 2000 / Accepted: 29 September 2000 / Published online: 11 November 2000 Ó Springer-Verlag 2000
Abstract The SeqA protein acts as a regulator of chromosomal replication initiation in Escherichia coli by sequestering hemi-methylated oriC, eectively blocking methylation and therefore preventing rapid re-initiation. The level of SeqA protein is maximal at mid-log phase and decreases when cells enter late-log phase. In hup mutants that lack the HU protein, the maximal seqA expression is also seen at mid-log phase, but seqA expression, as well as SeqA levels and activity, is increased by up to four fold relative to that in the wild type. These results suggest that the HU protein functions as a negative modulator of seqA expression. Key words
seqA á HU protein
Introduction In Escherichia coli, initiation of chromosomal replication occurs at oriC, the origin of chromosomal replication (Marsh and Worcel 1977; von Meyenburg et al. 1978), a 245-bp sequence that contains 11 GATC repeats (Zyskind and Smith 1986). GATC sites at oriC are methylated on the 6-amino group of the adenine residue by Dam methylase (Geier and Modrich 1979). Until methylated by Dam methylase, newly replicated GATC sequences remain hemimethylated, i.e. the template strand is methylated and the nascent strand is not (Campbell and Kleckner 1990). Conversion of the hemimethylated oriC to the fully methylated form by Dam methylase is delayed for one third of the cell cycle, relative to methylation at non-oriC GATC sites (Campbell Communicated by H. Ikeda H. Lee á H. K. Kim á S. Kang á C. B. Hong J. Yim á D. S. Hwang (&) Institute for Molecular Biology and Genetics, School of Biological Sciences, Seoul National University, Seoul 151-742, Korea Tel.: +82-2-880-7524 Fax: +82-2-874-1206
and Kleckner 1990; Lu et al. 1994). This is due to the preferential binding of the SeqA protein, encoded by the seqA gene, to the hemimethylated oriC (Campbell and Kleckner 1990; Lu et al. 1994; Slater et al. 1995; Brendler and Austin 1999; Kang et al. 1999). Null mutants for seqA exhibit asynchronous and increased initiation of chromosomal replication, indicating that SeqA is a negative modulator of chromosomal replication (Lu et al. 1994; von Freiesleben et al. 1994; Boye et al. 1996). The SeqA protein is therefore able to inhibit replication initiation at oriC, as well as preventing methylation of hemimethylated oriC. Intracellular localization of SeqA protein using immuno¯uorescence microscopy and a SeqA-GFP fusion indicates that SeqA forms foci on chromosomal DNA, and the foci are duplicated in a cell-cycle dependent manner (Hiraga et al. 1998; Onogi et al. 1999). Two foci migrate away from midcell, while two remain tethered in the mid-cell region until the FtsZ ring forms for cell division. SeqA foci also appear to be involved in segregation of the chromosomes. Fluorescence in
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