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

Afonso, Jos� P.; Chintakayala, Kiran; Suwannachart, Chatrudee; Sedelnikova, Svetlana; Giles, Kevin; Hoyes, John B.; Soultanas, Panos; Rafferty, John B.; Oldham, Neil J.

Abstract

The clamp-loader complex plays a crucial role in DNA replication by loading the β-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 τ3-δ-δ’ Bacillus subtilis clamp-loader assembles via multiple pathways,
which differ from those exhibited by the Gram-negative model E. coli. Based on this information a
homology model of the Bacillus subtilis τ3-δ-δ' complex was constructed, which revealed the spatial
positioning of the full C-terminal τ domain. The structure of the δ 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 τ and δ' binding regions. Most notably, the τ-δ interaction appears to be hydrophilic in nature
compared to the hydrophobic interaction in E. coli. Finally, the interaction between τ3 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 stabilise its
interaction with τ3.

Citation

Afonso, J. P., Chintakayala, K., Suwannachart, C., Sedelnikova, S., Giles, K., Hoyes, J. B., …Oldham, N. J. (2013). Insights into the structure and assembly of the Bacillus subtilis clamp-loader complex and its interaction with the replicative helicase. Nucleic Acids Research, 41(9), https://doi.org/10.1093/nar/gkt173

• Journal Article Type: Article
• Publication Date: May 1, 2013
• Deposit Date: Jun 14, 2013
• Publicly Available Date: Jun 14, 2013
• Journal: Nucleic Acids Research
• Print ISSN: 0305-1048
• Electronic ISSN: 0305-1048
• Publisher: Oxford University Press
• Peer Reviewed: Peer Reviewed
• Volume: 41
• Issue: 9
• DOI: https://doi.org/10.1093/nar/gkt173
• Public URL: https://nottingham-repository.worktribe.com/output/713963
• Publisher URL: http://nar.oxfordjournals.org/content/41/9/5115