Dam methylated and hemimethylatedoriC plasmids are replicated symmetrically; a novel and general test of replication symmetry
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Deoxyadenosine methylation (dam) of the numerous GATC sequences present in theEscherichia coli origin of chromosomal replication (oriC) has been shown to be important both in vivo and in vitro for efficient initiation of DNA synthesis. Recent in vivo data suggest that initiation is only inefficient when these sequences are hemimethylated. This raises the interesting possibility that initiation may be inefficient because it only takes place on one strand of the template, i.e., replication is asymmetric on hemimethylated DNA. We tested this possibility by a novel and rapid approach which relies on the specificities of the restriction endonucleasesMboI,MboII andDpnI. Although we show that replication takes place equally well on both strands of methylated and hemimethylatedoriC DNA templates, the method should be applicable to the analysis of replication symmetry on most DNA templates which contain methylated deoxyadenosine GATC sequences as part ofMboII restriction sites.
Key wordsReplication symmetry Restriction enzymes oriC GATC sequences Hemimethylation
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