NOVEL CYTOPLASMIC HEMIMETHYLATED ORIC BINDING-ACTIVITY

Using hemimethylated, fully methylated, and unmethylated oligonucleoti de probes corresponding to part of the origin of Escherichia coli DNA replication, oriC (+81-136), we have characterized a novel hemimethyla ted DNA-specific protein binding activity. This activity appears to be located in the cytoplasm rather than in membrane fractions. It has be en partially purified and, in DNase footprinting analysis, found to pr eferentially protect only a subset of the hemimethylated GATC sites pr esent 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 prot eins, although in the seqA deletion mutant, there is a 3-fold reductio n of the activity. The stage of the cell cycle in synchronized PC2 cul tures does not seem to significantly affect the relative levels of thi s binding activity. A possible role in sequestration of the newly repl icated hemimethylated origin is discussed.