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

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), Article 160272. 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: The Royal Society
• 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: https://nottingham-repository.worktribe.com/output/832662
• Publisher URL: http://rsob.royalsocietypublishing.org/content/6/12/160272