Cell cycle-specific changes in nucleoprotein complexes at a chromosomal replication origin.

Initiation of DNA synthesis is triggered by the binding of proteins to replication origins. However, little is known about the order in which specific proteins associate with origin sites during the cell cycle. We show that in cycling cells there are at least two different nucleoprotein complexes at oriC. A factor for inversion stimulation (FIS)-bound nucleoprotein complex, present throughout the majority of the cell cycle, switches to an integration host factor (IHF)-bound form as cells initiate DNA replication. Coincident with binding of IHF, initiator DnaA binds to its previously unoccupied R3 site. In stationary phase, a third nucleoprotein complex forms. FIS is absent and inactive oriC forms a nucleoprotein structure containing IHF that is not observed in cycling cells. We propose that interplay between FIS and IHF aids assembly of initiation nucleoprotein complexes during the cell cycle and blocks initiation at inappropriate times. This exchange of components at replication origins is reminiscent of switching between pre- and post-replicative chromatin states at yeast ARS1.     

Studies on the binding of integration host factor (IHF) and TraM to the origin of transfer of the IncFV plasmid pED208

The origin of transfer (oriT) of the IncFV plasmid pED208 contains a region with three binding sites for both the plasmid-encoded TraM protein and the integration host factor (IHF) of Escherichia coli, a sequence-specific DNA-binding protein. One region, containing overlapping TraM and IHF binding sites, could be interpreted as containing two binding sites for each protein. Using gel retardation assays, an affinity constant for IHF binding to the three main sites was estimated in the presence and absence of 0.1 M potassium glutamate, which increased the avidity of IHF binding to the weaker sites by two orders of magnitude. DNase I protection analyses and electron microscopy were used to determine the affinity of IHF for oriT-containing DNA in the presence and absence of TraM. The binding of IHF and TraM was found to be non-cooperative by the two techniques employed. Electron microscopy also demonstrated that IHF bent the oriT region in a manner consistent with its previously determined mode of action, while TraM had no discernible effect on the appearance of the DNA. This suggested that IHF and TraM interact with a 295 by sequence in the oriT region and organize it into a higher order structure that may have a role in the initiation of DNA transfer and control of traM expression.