SeqA proteins of Escherichia coli bound to the hemimethylated GATC sequences (hemi-sites) interact with each other and eventually form an aggregate. SeqA foci, which are suggested to be formed by aggregation, play important roles in the regulation of chromosome replication and segregation. We found that aggregation of SeqA proteins was preceded by cooperative interactions between these proteins bound to hemi-sites. Positively charged amino acids in the hinge region, which connects the N-terminal and C-terminal domain of SeqA, were critical for SeqA aggregation on hemimethylated DNA. Although the substitution of positively charged amino acids with negatively charged or neutral amino acids maintained the binding and cooperative interaction of mutant proteins, these proteins were defective in aggregation and foci formation in vitro and in vivo, respectively. Our results suggest that in vivo SeqA foci were formed by aggregation following cooperative interactions between SeqA proteins bound to hemi-sites. (c) 2007 Elsevier Inc. All rights reserved.