Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile
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. Clost...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Royal Society Publishing
2016
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| Online Access: | https://eprints.nottingham.ac.uk/44719/ |
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| author | van Eijk, Erika Paschalis, Vasileios Green, Matthew Friggen, Annemieke H. Larson, Marilynn A. Spriggs, Keith Briggs, Geoffrey S. Soultanas, Panos Smits, Wiep Klaas |
| author_facet | van Eijk, Erika Paschalis, Vasileios Green, Matthew Friggen, Annemieke H. Larson, Marilynn A. Spriggs, Keith Briggs, Geoffrey S. Soultanas, Panos Smits, Wiep Klaas |
| author_sort | van Eijk, Erika |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-14T19:56:38Z |
| format | Article |
| id | nottingham-44719 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T19:56:38Z |
| publishDate | 2016 |
| publisher | Royal Society Publishing |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-447192020-05-08T11:30:54Z https://eprints.nottingham.ac.uk/44719/ 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 Klaas 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. Royal Society Publishing 2016-12-21 Article PeerReviewed application/pdf en cc_by https://eprints.nottingham.ac.uk/44719/1/Open%20Biology%20WKS%20rsob-6-160272.pdf van Eijk, Erika, Paschalis, Vasileios, Green, Matthew, Friggen, Annemieke H., Larson, Marilynn A., Spriggs, Keith, Briggs, Geoffrey S., Soultanas, Panos and Smits, Wiep Klaas (2016) Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile. Open Biology, 6 (12). 160272/1-160272/16. ISSN 2046-2441 DNA replication initiation helicase loading and activation primase trinucleotide specificity ATPase Clostridium difficile http://rsob.royalsocietypublishing.org/content/6/12/160272 doi:10.1098/rsob.160272 doi:10.1098/rsob.160272 |
| spellingShingle | DNA replication initiation helicase loading and activation primase trinucleotide specificity ATPase Clostridium difficile van Eijk, Erika Paschalis, Vasileios Green, Matthew Friggen, Annemieke H. Larson, Marilynn A. Spriggs, Keith Briggs, Geoffrey S. Soultanas, Panos Smits, Wiep Klaas Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile |
| title | Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile |
| title_full | Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile |
| title_fullStr | Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile |
| title_full_unstemmed | Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile |
| title_short | Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile |
| title_sort | primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen clostridium difficile |
| topic | DNA replication initiation helicase loading and activation primase trinucleotide specificity ATPase Clostridium difficile |
| url | https://eprints.nottingham.ac.uk/44719/ https://eprints.nottingham.ac.uk/44719/ https://eprints.nottingham.ac.uk/44719/ |