title: Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile.
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.
authors.affiliation: Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Erika
authors.initials: E
authors.lastname: van Eijk
authors.affiliation: School of Chemistry, Center for Biomolecular Sciences, University of Nottingham, UK.
authors.forename: Vasileios
authors.identifier: 0000-0003-2757-6918
authors.initials: V
authors.lastname: Paschalis
authors.affiliation: School of Chemistry, Center for Biomolecular Sciences, University of Nottingham, UK.
authors.forename: Matthew
authors.identifier: 0000-0003-2771-9894
authors.initials: M
authors.lastname: Green
authors.affiliation: Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Annemieke H
authors.initials: AH
authors.lastname: Friggen
authors.affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198-5900, USA.|National Strategic Research Institute, Omaha, NE 68105, USA.
authors.forename: Marilynn A
authors.identifier: 0000-0002-0633-0313
authors.initials: MA
authors.lastname: Larson
authors.affiliation: School of Pharmacy, University of Nottingham, UK.
authors.forename: Keith
authors.identifier: 0000-0001-5946-1440
authors.initials: K
authors.lastname: Spriggs
authors.affiliation: School of Chemistry, Center for Biomolecular Sciences, University of Nottingham, UK.
authors.forename: Geoffrey S
authors.identifier: 0000-0002-4149-8752
authors.initials: GS
authors.lastname: Briggs
authors.affiliation: School of Chemistry, Center for Biomolecular Sciences, University of Nottingham, UK.
authors.forename: Panos
authors.identifier: 0000-0003-3578-5699
authors.initials: P
authors.lastname: Soultanas
authors.affiliation: Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands w.k.smits@lumc.nl.
authors.forename: Wiep Klaas
authors.identifier: 0000-0002-7409-2847
authors.initials: WK
authors.lastname: Smits
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016360:Clostridioides difficile / Q000201:enzymology / Q000235:genetics*; D003198:Computer Simulation; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019915:DNA Primase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics; D009154:Mutation; D011485:Protein Binding
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D000255:Adenosine Triphosphate; D019915:DNA Primase; D004265:DNA Helicases
keywords: ATPase; Clostridium difficile; DNA replication initiation; helicase loading and activation; primase trinucleotide specificity