Characterization of the role of stabilized DNA bending during pre-replicative complex assembly at Escherichia coli oriC

All cells must replicate their genomes before reproducing. So that each daughter cell receives identical genetic information, initiation of chromosome replication is tightly regulated by focusing control mechanisms on the assembly of a nucleoprotein pre-replicative complex (pre-RC) that unwinds origin DNA. One of the best characterized models for the assembly of this complex is in Eshcherichia coli (E. coli), where the pre-RC comprises the unique origin of replication (oriC) and 15-20 molecules of the initiator protein DnaA. Accessory factors regulate pre-RC assembly by modulating DnaA binding, and at least two of these factors, Factor for Inversion Stimulation (FIS), and Integration Host Factor (IHF), are known to place bends in DNA.;The goal of this study was to determine the role of DNA bending in pre-RC assembly. Using electrophoretic mobility shift assays, site directed mutagenesis, and dimethyl sulfate footprinting, we determined that a region of flexible DNA in oriC is normally stabilized by both IHF binding and by the cross-strand interaction of DnaA during pre-RC assembly. Cross-strand DnaA interaction, verified by cross-linking assays, formed a loop, and also promoted cooperative binding between a high and low affinity DnaA binding site. Mutations that promoted more efficient co-operative binding resulted in more efficient DNA strand separation, indicating that DnaA binding to sites in oriC's left half is a key feature in the mechanism of oriC unwinding. The loop was essential for oriC function, and disruption of one of the loop's stabilizing mechanisms by mutagenesis of chromosomal oriC caused cells to under-initiate chromosome replication, while loss of all stabilizing mechanisms resulted in loss of oriC function. Mutations that made oriC unbendable also eliminated chromosomal oriC function, even if cooperative binding was permitted. These results suggest that, in addition to mediating cooperative DnaA interactions, DNA bending also promotes a configuration that licenses subsequent stages of initiation. Both functions are integrated into a single pre-RC sub-complex. We propose that this sub-complex produces a DNA replication switch that is incorporated into a very short stretch of DNA, coordinating origin unwinding with the final stages of pre-RC assembly.