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

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

Authors

José P. Afonso

Kiran Chintakayala

Chatrudee Suwannachart

Svetlana Sedelnikova

Kevin Giles

John B. Hoyes

Panos Soultanas panos.soultanas@nottingham.ac.uk

John B. Rafferty

Neil J. Oldham

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.

Journal Article Type Article
Publication Date May 1, 2013
Journal Nucleic Acids Research
Print ISSN 0305-1048
Electronic ISSN 0305-1048
Publisher Oxford University Press (OUP)
Peer Reviewed Peer Reviewed
Volume 41
Issue 9
Institution 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), doi:10.1093/nar/gkt173
DOI https://doi.org/10.1093/nar/gkt173
Publisher URL http://nar.oxfordjournals.org/content/41/9/5115
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by-nc/4.0

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Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by-nc/4.0




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