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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...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Royal Society
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5204125/ https://www.ncbi.nlm.nih.gov/pubmed/28003473 http://dx.doi.org/10.1098/rsob.160272 |
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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 |
collection | PubMed |
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. |
format | Online Article Text |
id | pubmed-5204125 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | The Royal Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-52041252017-01-05 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 Open Biol Research 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. The Royal Society 2016-12-21 /pmc/articles/PMC5204125/ /pubmed/28003473 http://dx.doi.org/10.1098/rsob.160272 Text en © 2016 The Authors. http://creativecommons.org/licenses/by/4.0/ Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. |
spellingShingle | Research 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 | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5204125/ https://www.ncbi.nlm.nih.gov/pubmed/28003473 http://dx.doi.org/10.1098/rsob.160272 |
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