Insights into the structure and assembly of the Bacillus subtilis clamp-loader complex and its interaction with the replicative helicase

摘要

The clamp-loader complex plays a crucial role in DNA replication by loading the beta-clamp onto primed DNA to be used by the replicative polymerase. Relatively little is known about the stoichiometry, structure and assembly pathway of this complex, and how it interacts with the replicative helicase, in Gram-positive organisms. Analysis of full and partial complexes by mass spectrometry revealed that a hetero-pentameric tau(3)-delta-delta' Bacillus subtilis clamp-loader assembles via multiple pathways, which differ from those exhibited by the Gram-negative model Escherichia coli. Based on this information, a homology model of the B. subtilis tau(3)-delta-delta' complex was constructed, which revealed the spatial positioning of the full C-terminal tau domain. The structure of the delta subunit was determined by X-ray crystallography and shown to differ from that of E. coli in the nature of the amino acids comprising the tau and delta' binding regions. Most notably, the tau-delta interaction appears to be hydrophilic in nature compared with the hydrophobic interaction in E. coli. Finally, the interaction between tau(3) and the replicative helicase DnaB was driven by ATP/Mg2+ conformational changes in DnaB, and evidence is provided that hydrolysis of one ATP molecule by the DnaB hexamer is sufficient to stabilize its interaction with tau(3).