Nucleotide Control of Replication Initiation: NTPase Mechanisms of E.coli DnaB-DnaC Complex

Abstract

In E. coli, interactions between the primary replicative helicase, DnaB protein and the replication factor, DnaC protein, are at the heart of the chromosomal DNA replication. To address the role of the DnaC in processes of the free energy transduction by the helicase, we have quantitatively examined DnaB-DnaC complex using fluorescence titration, analytical ultracentrifugation, and rapid chemical quench-flow techniques.In absence of nucleic acid, DnaC reduces intrinsic affinity and increases negative cooperativity in nucleotide binding to DnaB helicase. The ground-state effects are accompanied by the reduced rate of ATP hydrolysis by the helicase. In presence of DNA, DnaB in the DnaB-DnaC complex recovers its nucleotide binding capabilities and ATPase activity. These data suggest that recognition of the oriC by the DnaB - DnaC complex and/or its entry into the pre-primosome requires diminished NTPase activity of the helicase. Analysis of nucleotide binding to the DnaC protein, engaged in the DnaB - DnaC complex, indicates that prior to the recognition of the oriC sequence and/or pre-primosome assembly, the DnaC protein in the complex is in a conformational state, which does not bind ATP or ADP. So, the formation of the replisome and the pre-primosome seems to preferentially require the presence of DnaC in a state free of cofactors. Significant positive cooperativity of the binding process indicates that small fluctuations in ATP and/or ADP concentrations can induce an all-or-none allosteric transition of bound DnaC molecules into the conformation, which has an increased intrinsic affinity for the nucleotides. The presence of such an all-or-none allosteric transition, encompassing all bound DnaC molecules, indicates that recognition of the oriC and the pre-primosome assembly includes complex interplay between different conformations of the DnaB - DnaC complex. The physiological importance of the obtained results will be discussed.