Sequential binding of SeqA to paired hemi-methylated GATC sequences mediates formation of higher order complexes

Preferential binding of the SeqA protein to hemi-methylated GATC sequences functions as a negative regulator for Escherichia coli initiation of chromosomal replication at oriC and is implicated in segregating replicated chromosomes for cell division. We demonstrate that sequential binding of one SeqA tetramer to a set of two hemi-methylated sites mediates formation of higher-order complexes. The absence of cross-binding to separate DNAs suggests that two monomers of a SeqA tetramer bind to two hemi-methylated sites on DNA. The interaction among SeqA proteins bound to at least six adjacent hemi-methylated sites induces aggregation of free proteins to bound proteins. Aggregation might be indicative of SeqA foci, which appear to track replication forks in vivo. Studies of the properties of SeqA binding will contribute to our understanding of the function of SeqA.