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 β-clamp onto primedudDNA to be used by the replicative polymerase. Relatively little is known about the stoichiometry,udstructure and assembly pathway of this complex, and how it interacts with the replicative helicase,udin Gram-positive organisms. Analysis of full and partial complexes by mass spectrometry revealedudthat a hetero-pentameric τ3-δ-δ’ Bacillus subtilis clamp-loader assembles via multiple pathways,udwhich differ from those exhibited by the Gram-negative model E. coli. Based on this information audhomology model of the Bacillus subtilis τ3-δ-δ' complex was constructed, which revealed the spatialudpositioning of the full C-terminal τ domain. The structure of the δ subunit was determined by X-rayudcrystallography and shown to differ from that of E. coli in the nature of the amino acids comprisingudthe τ and δ' binding regions. Most notably, the τ-δ interaction appears to be hydrophilic in natureudcompared to the hydrophobic interaction in E. coli. Finally, the interaction between τ3 and theudreplicative helicase DnaB was driven by ATP/Mg2+ conformational changes in DnaB and evidence isudprovided that hydrolysis of one ATP molecule by the DnaB hexamer is sufficient to stabilise itsudinteraction with τ3.