Dam methylated and hemimethylated oriC plasmids are replicated symmetrically; a novel and general test of replication symmetry.
Deoxyadenosine methylation (dam) of the numerous GATC sequences present in the Escherichia 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 endonucleases MboI, MboII and DpnI. Although we show that replication takes place equally well on both strands of methylated and hemimethylated oriC 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 of MboII restriction sites.
Hughes
P
P
Institut Jacques Monod, Centre National de la Recherche Scientifique, Université Paris, France.
Landoulsi
A
A
Kern
R
R
Kohiyama
M
M
D001483:Base Sequence; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D003839:Deoxyadenosines / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D010957:Plasmids
D004269:DNA, Bacterial; D003839:Deoxyadenosines