Lack of SeqA focus formation, specific DNA binding and proper protein multimerization in the Escherichia coli sequestration mutant seqA2.
In Escherichia coli wild-type cells newly formed origins cannot be reinitiated. The prevention of reinitiation is termed sequestration and is dependent on the hemimethylated state of newly replicated DNA. Several mutants discovered in a screen for the inability to sequester hemimethylated origins have been mapped to the seqA gene. Here, one of these mutants, seqA2, harbouring a single amino acid change in the C-terminal end of the SeqA protein, was found to also be unable to form foci in vivo. The SeqA foci seen in the wild-type cells are believed to arise from multimerization of SeqA on hemimethylated DNA at the replication fork, presumably representing organization of newly formed DNA by SeqA. The result suggests that the process of origin sequestration is closely tied to the process of focus maintenance at the replication fork. In vitro, purified SeqA2 protein was found incapable of forming highly ordered multimers that bind hemimethylated oriC. The mutant protein was also incapable of restraining negative supercoils. Both in vivo and in vitro results support the idea that origin sequestration is an integral part of organization of newly formed DNA performed by SeqA.
Fossum
Solveig
S
Department of Cell Biology, Institute for Cancer Research, Montebello, 0310 Oslo, Norway.
Søreide
Sølvi
S
Skarstad
Kirsten
K
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D009154:Mutation; D051738:Origin Recognition Complex; D018741:Replication Origin; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; C114650:OriC chromosomal replication origin; D051738:Origin Recognition Complex; C089746:SeqA protein, E coli; D014157:Transcription Factors; D014764:Viral Proteins