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Nucleic Acids Research, 1992, Vol. 20, No. 1 105-109
© 1992

MOLECULAR BIOLOGY

Inhibition of DNA synthesis at the hemimethylated pBR322 origin of replication by a cell membrane fraction

Abderrahim Malki, Renée Kern, Masamichi Kohiyama and Patrick Hughes*

Department of Molecular Biology, Institut Jacques Monod Tour 43, Universite Paris VII, 2 Place Jussieu, 75251 Paris Cedex 05, France

* To whom correspondence should be addressed

Received September 12, 1991. Revised December 9, 1991. Accepted December 9, 1991.

The replication of both ColE1-type plasmids and plasmids bearing the origin of replication of the Escherichia coli chromosome (oriC) has been shown within GATC sequences. In the case of oriC plasmids, this inhibition was previously shown to be mediated by the specific affinity of the hemimethylated origin DNA for an outer cell membrane fraction. Here, we suggest that a similar mechanism is operating in the case of the ColE1-Iike plasmid pBR322 as (i) a hemimethylated DNA fragment carrying the promoter for the [RNA which primes DNA synthesis (RNAll) is specifically bound by the same membrane fraction and, (ii) the addition of the membrane fraction to a soluble assay of pBR322 replication results in preferential inhibition of initiation on the hemimethylated template. We suggest that membrane sequestration of hemimethylated origin DNA and/or associated replication genes following replication may be a common element restricting DUMA replication to precise moments in the cell cycle.


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