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Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile

van Eijk, Erika; Paschalis, Vasileios; Green, Matthew; Friggen, Annemieke H.; Larson, Marilynn A.; Spriggs, Keith; Briggs, Geoffrey S.; Soultanas, Panos; Smits, Wiep Klass

Authors

Erika van Eijk

Vasileios Paschalis

Matthew Green

Annemieke H. Friggen

Marilynn A. Larson

Geoffrey S. Briggs

Wiep Klass Smits



Abstract

DNA replication is an essential and conserved process in all domains of life and may serve as a target for the development of new antimicrobials. However, such developments are hindered by subtle mechanistic differences and limited understanding of DNA replication in pathogenic microorganisms. Clostridium difficile is the main cause of healthcare-associated diarrhoea and its DNA replication machinery is virtually uncharacterized. We identify and characterize the mechanistic details of the putative replicative helicase (CD3657), helicase-loader ATPase (CD3654) and primase (CD1454) of C. difficile, and reconstitute helicase and primase activities in vitro We demonstrate a direct and ATP-dependent interaction between the helicase loader and the helicase. Furthermore, we find that helicase activity is dependent on the presence of primase in vitro The inherent trinucleotide specificity of primase is determined by a single lysine residue and is similar to the primase of the extreme thermophile Aquifex aeolicus. However, the presence of helicase allows more efficient de novo synthesis of RNA primers from non-preferred trinucleotides. Thus, loader-helicase-primase interactions, which crucially mediate helicase loading and activation during DNA replication in all organisms, differ critically in C. difficile from that of the well-studied Gram-positive Bacillus subtilis model.

Citation

van Eijk, E., Paschalis, V., Green, M., Friggen, A. H., Larson, M. A., Spriggs, K., …Smits, W. K. (2016). Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile. Open Biology, 6(12), https://doi.org/10.1098/rsob.160272

Journal Article Type Article
Acceptance Date Nov 22, 2016
Online Publication Date Dec 21, 2016
Publication Date Dec 21, 2016
Deposit Date Aug 7, 2017
Publicly Available Date Aug 7, 2017
Journal Open Biology
Electronic ISSN 2046-2441
Publisher Royal Society, The
Peer Reviewed Peer Reviewed
Volume 6
Issue 12
Article Number 160272
DOI https://doi.org/10.1098/rsob.160272
Keywords DNA replication initiation, helicase loading and activation, primase trinucleotide specificity, ATPase, Clostridium difficile
Public URL http://eprints.nottingham.ac.uk/id/eprint/44719
Publisher URL http://rsob.royalsocietypublishing.org/content/6/12/160272
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/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/4.0





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