Periodic formation of the oriC complex of Escherichia coli.

Abstract

We examined formation of an oriC‐membrane complex through the chromosome replication cycle by dot‐blot hybridization using an oriC plasmid as a probe. In a wild‐type culture synchronized for chromosome replication, oriC complex formation was observed periodically and transiently corresponding to the replication initiation event. Prior to initiation of replication the oriC complex was recovered in the outer membrane fraction as well as at the time of initiation of replication. Moreover, periodic formation of the oriC complex was observed even when further initiation of replication was suppressed by culturing an initiation ts mutant at the restrictive temperature. Similar periodic formation of the oriC complex was also observed when DNA elongation was inhibited by addition of nalidixic acid to the culture. However, the second periodic peak did not appear when rifampicin or chloramphenicol was added. Cells which formed the oriC complex at the restrictive temperature could immediately initiate chromosome replication when the cells were transferred to the permissive temperature. We conclude that the oriC region of Escherichia coli forms a specific complex periodically just before and at the time of initiation of chromosome replication and that oriC complex formation is a prerequisite for initiation of chromosome replication.

Simian virus 40 T-antigen DNA helicase is a hexamer which forms a binary complex during bidirectional unwinding from the viral origin of DNA replication

“…Moreover, the DNA helicase could function as an organizer of the replication machine and at the same time could fix it to the nuclear scaffold, possibly arranged in replication centers with multiple replication forks as has been suggested recently for viral and cellular DNA (13,24,34,54). Accordingly, the (transient) binding of replication origins, as well as replicating DNA, to structural elements has been described (18,21,26,55; for a review, see reference 53), and during a lytic infection, a subfraction of T antigen is bound to the nuclear matrix and has been suggested to be actively involved in SV40 DNA replication (42,43,57). In this context, it is also interesting to note the frequent colocalization of detected replication origins with matrix attachment sites (1-3, 9).…”

A Novel Cytoplasmic Hemimethylated oriC Binding Activity

“…Many studies of oriC membrane-associated interactions have employed such a preparative procedure, fractionating the lysates obtained after passage through a French pressure cell by ultracentrifugation of sucrose gradients (6,8,9,16,19,20). Origin DNA has been found in membrane fractions that were prepared in this manner, but its presence seems to vary according to the initiation potential of the cell as shown by the use of synchronized cultures of initiation mutants (19,23). Hence, these studies have also employed synchronized cultures of the thermosensitive dnaC mutant, PC2 (17), in which, at the nonpermissive growth temperature (Ͼ37°C), new rounds of initiation of DNA replication are blocked while those that have begun continue until terminated; by bringing a culture of PC2 back to the permissive temperature (Ͻ36°C) after 1 h at the nonpermissive temperature there is a synchronized reinitiation of all origins in the culture (24).…”

Anucleate cell production by Escherichia coli delta hns mutant lacking a histone-like protein, H-NS

“…The H-NS protein was detected in oriC-membrane complex fractions by using an anti-H-NS antibody (data not shown). The oriC-membrane complex is formed just before and after initiation (10,15) and may function in sequestration of the next round of initiation at oriC. Recently, isolation of a mutant defective in this step, seqA, was reported (18).…”