By means of a nitrocellulose filter binding assay, DNA binding activities among proteins fractionated from extracts of Escherichia coli carrying 2dv have been surveyed. An activity was found that binds specifically to a fragment of 164 base pairs that specifies the 2 replication origin (2 ori). This activity was not detected in an extract of cells not carrying the ~,dv plasmid. The activity was detected in extracts of cells carrying a hybrid plasmid in which the entire )~ O gene had been cloned and placed under the control of the lac promoter. Deletion of a 60 base pair segment in the 'amino-terminal region' of the O gene abolished this activity, indicating that the 2 ori binding protein is coded for by the 20 gene.
The or#specific binding protein was purified by five fractionation steps. The most purified preparation consists of a major polypeptide that migrates with a molecular weight of 32,000 in SDS-polyacrylamide gel electrophoresis. Binding of O protein to ori occurs in the absence of other protein aceous components.
that it is the amino-terminal domain of this protein that interacts with ori, whereas the carboxyl-terminal domain interacts with P protein. Despite its important role in DNA initiation, O protein has so far not been purified. To achieve this, two fractors were taken into consideration: (i) Its function cannot be detected in the current in vitro complementation assay system for 2 DNA synthesis (Klein et al. 1978), therefore, the )~ ori binding activity was taken as a measure of the activity of O protein; (ii) There is little O protein in the cell, possibly due to its short half-life (Lipinska et al. 1980 ; Miwa and Akaboshi personal communication). We took as starting material E. colicells carrying a promoter-mutant 2dv or a recombinant plasmid in which the O gene had been inserted. Because such cells contain many copies of the plasmid and because the O gene is expressed with high efficiency in the promoters utilized, the cellular level of O protein was expected to be high, despite its instability.
This paper describes purification of the O protein and some of its properties.