SeqA protein aggregation is necessary for SeqA function.
The binding of SeqA protein to hemimethylated GATC sequences is important in the negative modulation of chromosomal initiation at oriC, and in the formation of SeqA foci necessary for Escherichia coli chromosome segregation. Using gel-filtration chromotography and glycerol gradient sedimentation, we demonstrate that SeqA exists as a homotetramer. SeqA tetramers are able to aggregate or multimerize in a reversible, concentration-dependent manner. Using a bacterial two-hybrid system, we demonstrate that the N-terminal region of SeqA, especifically the 9th amino acid residue, glutamic acid, is required for functional SeqA-SeqA interaction. Although the SeqA(E9K) mutant protein, containing lysine rather than glutamic acid at the 9th amino acid residue, exists as a tetramer, the mutant protein binds to hemimethylated DNA with altered binding patterns as compared with wild-type SeqA. Aggregates of SeqA(E9K) are defective in hemimethylated DNA binding. Here we demonstrate that proper interaction between SeqA tetramers is required for both hemimethylated DNA binding and formation of active aggregates. SeqA tetramers and aggregates might be involved in the formation of SeqA foci required for the segregation of chromosomal DNA as well as the regulation of chromosomal initiation.
Lee
H
H
Institute of Molecular Biology and Genetics, School of Biological Sciences, Seoul National University, Seoul 151-742, Korea.
Kang
S
S
Bae
S H
SH
Choi
B S
BS
Hwang
D S
DS
D001425:Bacterial Outer Membrane Proteins; D004247:DNA / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D014157:Transcription Factors / Q000737:chemistry* / Q000502:physiology; D020798:Two-Hybrid System Techniques
D001425:Bacterial Outer Membrane Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C089746:SeqA protein, E coli; D014157:Transcription Factors; D004247:DNA