title: P1 plasmid replication requires methylated DNA.
abstract: Plasmids driven by the plasmid replication origin of bacteriophage P1 cannot be established in Escherichia coli strains that are defective for the DNA adenine methylase (dam). Using a composite plasmid that has two origins, we show that the P1 origin cannot function even in a plasmid that is already established in a dam strain. An in vitro replication system for the P1 origin was developed that uses as a substrate M13 replicative-form DNA containing the minimal P1 origin. The reaction mixture contains a crude extract of E. coli and purified P1 RepA protein. In addition to being RepA dependent, synthesis was shown to be dependent on methylation of the dam methylase-sensitive sites of the substrate DNA. As the P1 origin contains five such sites in a small region known to be critical for origin function, it can be concluded that methylation of these sites is a requirement for initiation. This suggests that the postreplicational methylation of the origin may control reinitiation and contribute to the accuracy of the highly stringent copy-number control of the origin in vivo.
authors.affiliation: Laboratory of Chromosome Biology, BRI-Basic Research Program, NCI-Frederick Cancer Research Facility, MD 21701.
authors.forename: A L
authors.initials: AL
authors.lastname: Abeles
authors.forename: S J
authors.initials: SJ
authors.lastname: Austin
mesh_terms: D001483:Base Sequence; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)
chemical_list: D004269:DNA, Bacterial; D008780:Methyltransferases; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D004262:DNA Restriction Enzymes