E. coli SeqA protein binds oriC in two different methyl-modulated reactions appropriate to its roles in DNA replication initiation and origin sequestration.
The seqA gene negatively modulates replication initiation at the E. coli origin, oriC. seqA is also essential for sequestration, which acts at oriC and the dnaA promoter to ensure that replication initiation occurs exactly once per chromosome per cell cycle. Initiation is promoted by full methylation of GATC sites clustered in oriC; sequestration is specific to the hemimethylated forms generated by replication. SeqA protein purification and DNA binding are described. SeqA interacts with fully methylated oriC strongly and specifically. This reaction requires multiple molecules of SeqA and determinants throughout oriC, including segments involved in open complex formation. SeqA interacts more strongly with hemimethylated DNA; in this case, oriC and non-oriC sequences are bound similarly. Also, binding of hemimethylated oriC by membrane fractions is due to SeqA. Direct interaction of SeqA protein with the replication origin is likely to be involved in both replication initiation and sequestration.
Slater
S
S
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Wold
S
S
Lu
M
M
Boye
E
E
Skarstad
K
K
Kleckner
N
N
D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004586:Electrophoresis; D004926:Escherichia coli / Q000166:cytology*; D029968:Escherichia coli Proteins; D008745:Methylation; D008969:Molecular Sequence Data; D018741:Replication Origin / Q000502:physiology*; D013379:Substrate Specificity; D014157:Transcription Factors
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C089746:SeqA protein, E coli; D014157:Transcription Factors