Replication of lambda dv plasmid in vitro promoted by purified lambda O and P proteins.

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Abstract
An in vitro system for replication of .lambda. dv plasmid DNA was constructed. This system consists of an ammonium sulfate fraction from Escherichia coli extract, exogenously added purified .lambda. O and P proteins, and .lambda. dv DNA in closed circular form. More than 85% of the added template DNA replicated semiconservatively. In the same system, another plasmid, pBR322, also replicated, but less efficiently than .lambda. dv. Its replication was independent of O and P proteins. Inhibitors of DNA gyrase entirely blocked the replication activity, whereas rifampicin, an inhibitor of RNA polymerase, showed a significant effect only when added prior to initiation of the DNA replication. DNA replication was initiated from a region near to or within the 4 direct repeats in .lambda. origin (.lambda. ori) and proceeded bidirectionally, as examined by DNA chain elongation termination with dideoxy CTP. A cloned DNA carrying a 350-base-pair region including the initiation site also initiated replication, dependent on O and P proteins, and its initiation occurred at the same positon as with native .lambda. dv DNA. An A + T-rich structure neighboring the repeats is essential for .lambda. DNA replication. Regions corresponding to ice and oop were not required for O,P-dependent initiation.