A novel cytoplasmic hemimethylated oriC binding activity.
Using hemimethylated, fully methylated, and unmethylated oligonucleotide probes corresponding to part of the origin of Escherichia coli DNA replication, oriC (+81-136), we have characterized a novel hemimethylated DNA-specific protein binding activity. This activity appears to be located in the cytoplasm rather than in membrane fractions. It has been partially purified and, in DNase footprinting analysis, found to preferentially protect only a subset of the hemimethylated GATC sites present in the minimal oriC. These sites are found adjacent to the DnaA binding box, R1, and overlap the integration host factor binding site. The activity does not correspond to known hemimethylated binding proteins, although in the seqA deletion mutant, there is a 3-fold reduction of the activity. The stage of the cell cycle in synchronized PC2 cultures does not seem to significantly affect thte relative levels of this binding activity. A possible role in sequestration of the newly replicated hemimethylated origin is discussed.
Garwood
J
J
Biochemical Genetics Group, Institut Jacques Monod, Université Paris, France.
Kohiyama
M
M
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003593:Cytoplasm / Q000378:metabolism; D018983:DNA Footprinting; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003851:Deoxyribonucleases; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015345:Oligonucleotide Probes; D018741:Replication Origin; D017384:Sequence Deletion; D014157:Transcription Factors
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; D015345:Oligonucleotide Probes; C089746:SeqA protein, E coli; D014157:Transcription Factors; D003851:Deoxyribonucleases