Specific N-terminal interactions of the Escherichia coli SeqA protein are required to form multimers that restrain negative supercoils and form foci.
The Escherichia coli SeqA protein binds preferentially to hemimethylated DNA and is required for inactivation (sequestration) of newly formed origins. A mutant SeqA protein, SeqA4 (A25T), which is deficient in origin sequestration in vivo, was found here to have lost the ability to form multimers, but could bind as dimers with wild-type affinity to a pair of hemimethylated GATC sites. In vitro, binding of SeqA dimers to a plasmid first generates a topology change equivalent to a few positive supercoils, then the binding leads to a topology change in the "opposite" direction, resulting in a restraint of negative supercoils. Binding of SeqA4 mutant dimers produced the former effect, but not the latter, showing that a topology change equivalent to positive supercoiling is caused by the binding of single dimers, whereas restraint of negative supercoils requires multimerization via the N-terminus. In vivo, mutant SeqA4 protein was not capable of forming foci observed by immunofluorescence microscopy, showing that N-terminus-dependent multimerization is required for building SeqA foci. Overproduction of SeqA4 led to partially restored initiation synchrony, indicating that origin sequestration may not depend on efficient higher-order multimerization into foci, but do require a high local concentration of SeqA.
Odsbu
Ingvild
I
Department of Cell Biology, Institute for Cancer Research, Montebello, 0310 Oslo, Norway.
Klungsøyr
Hege K
HK
Fossum
Solveig
S
Skarstad
Kirsten
K
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D019175:DNA Methylation; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D021381:Protein Transport; D018741:Replication Origin / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C089746:SeqA protein, E coli