Purified Rep (or RepA) protein, a replication initiator of plasmid pSC101, is present almost solely in the dimer form, and its binding activity for the directly repeated sequences (iterons) in the replication origin (
ori) is very low. When Rep protein was treated with guanidine hydrochloride followed by renaturation, it was shown to bind to the iterons with very high efficiency. A gel shift experiment suggested that guanidine-treated Rep bound to iterons as a monomer form. The Rep monomer bound noncooperatively to the three iterons and induced bending of the DNA helix axis in the same direction (about 100°). The configuration of the IHF box that is a binding site of another DNA bending protein IHF, the three iterons and an AT-rich region between these sequences was important for efficient bending of the
ori region. Furthermore, a mutant Rep protein (Rep
IHF) which can support the plasmid replication in IHF-deficient host cells was purified, and it was found that affinity of the Rep
IHF monomer for iterons was similar to that of wild-type Rep and bent DNA only 14° more strongly than did the wild-type Rep. Rep
IHF-dependent plasmid replication, however, required both enhancer regions, par and IR-1, in addition to "core
ori" as a minimal essential
ori, whereas only one of these two enhancers was necessary for wild-type Rep-dependent replication. How Rep
IHF can support plasmid replication in the absence of IHF is discussed.
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