Abstract
Origin opening is essential for the initiation of DNA replication in the theta mode and requires binding of initiator proteins. Using reactivity to KMnO4 in vivo as an assay, we find that, like initiation, origin opening of the Escherichia coli plasmid P1 requires the host initiators DnaA and HU and the plasmid-encoded initiator RepA. The ability to detect opening at the P1ori in vivo allowed us to study this activity at various copy numbers in chimeric replicons. The opening was prevented when the P1ori was cloned in high copy vectors or when excess RepA binding sites (iterons) were provided in trans. However, when RepA supply was also increased, the opening was efficient. A further increase in RepA prevented opening. Replication of an incoming P1 under these conditions correlated with opening. These results demonstrate that initiation is possible even at abnormally high origin concentrations and that oversupply of RepA, relative to iterons, can prevent replication by blocking origin opening. It appears that plasmid overreplication can be prevented either by limiting RepA or by accumulating RepA at a rate higher than that of the origin.
Highlights
We find that the requirements for the appearance of KMnO4reactive bases in P1ori are well correlated with the genetic determinants of P1 plasmid replication
Initiation of DNA replication has been staged into discrete steps primarily from the work in vitro on plasmids carrying the Escherichia coli origin, oriC
We have examined whether origin opening of plasmid P1 can be used to follow regulation of initiation in vivo
Summary
Plasmids, and Phages—Bacteria and their relevant genotypes were DH5⌬lac, recA [10]; PC2, dnaC2 [11]; EH3827, ⌬dnaA [4]; BR4587, hupA16 [12]; BR4586, hupB11 [12]; and BR4588, hupA16hupB11 (this study). Fragments containing the repA(ϮT1)bla-p2 region were cloned into a pBR322 compatible vector, pST52 [14] to generate plasmids pALA169, pALA198, and pALA197. Another 7ϫ RepA source was pKP116, constructed by cloning a SspI-EcoRV fragment of pALA176 [15] into the HincII site of pGB2, a pSC101-derived vector with a spectinomycin resistance gene [16]. KMnO4 was diluted to a final concentration of 3 mM and incubated for 1 min at 42 °C for all cells. The mixtures were incubated at 30 °C for 2 h for expression of drug resistance and, after appropriate dilution, plated on media selective for resident plasmids and infecting phage (plasmid prophage)
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