E. coli SeqA protein binds oriC in two different methyl-modulated reactions appropriate to its roles in DNA replication initiation and origin sequestration

• Published in: Cell Vol. 82; no. 6; pp. 927 - 936
• Main Authors: Slater, Steven, Wold, Sture, Lu, Min, Boye, Erik, Skarstad, Kirsten, Kleckner, Nancy
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 22.09.1995
• Subjects: Bacterial Outer Membrane Proteins - metabolism; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Base Sequence; Cell Membrane - metabolism; DNA Replication; DNA, Bacterial - biosynthesis; DNA, Bacterial - metabolism; DNA-Binding Proteins - metabolism; Electrophoresis; Escherichia coli; Escherichia coli - cytology; Escherichia coli Proteins; Methylation; Molecular Sequence Data; Replication Origin - physiology; Substrate Specificity; Transcription Factors
• ISSN: 0092-8674; 1097-4172
• DOI: 10.1016/0092-8674(95)90272-4

The segA gene negatively modulates replication initiation at the E. coil origin, oriC. segA 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. SegA protein purification and DNA binding are described. SegA interacts with fully methylated oriC strongly and specifically. This reaction requires multiple molecules of SegA and determinants throughout oriC, including segments involved in open complex formation. SegA 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 SegA. Direct interaction of SegA protein with the replication origin is likely to be involved in both replication initiation and sequestration.