DNA BENDING OF PSC101 ORI INDUCED BY REP, A REPLICATION INITIATOR PROTEIN AND IHF

Purified Rep (or RepA) protein, a replication initiator of plasmid pSC 101, is present almost solely in the dimer form, and its binding activ ity for the directly repeated sequences (iterons) in the replication o rigin (ori) is very low. When Rep protein was treated with guanidine h ydrochloride followed by renaturation, it was shown to bind to the ite rons with very high efficiency, A gel shift experiment suggested that guanidine-treated Rep bound to iterons as a monomer form, The Rep mono mer bound noncooperatively to the three iterons and induced bending of the DNA helix axis in the same direction (about 100 degrees), The con figuration of the IHF box that is a binding site of another DNA bendin g protein IHF, the three iterons and an AT-rich region between these s equences was important for efficient bending of the ori region, Furthe rmore, a mutant Rep protein (Rep(IHF)) which can support the plasmid r eplication 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 degrees more strongly than did t he wild-type Rep, Rep(IHF)-dependent plasmid replication, however, req uired both enhancer regions, par and IR-1, in addition to ''core ori'' as a minimal essential ori, whereas only one of these two enhancers w as necessary for wild-type Rep-dependent replication. How Rep(IHF) can support plasmid replication in the absence of IHF is discussed.