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DNA replication origins retain mobile licensing proteins
DNA replication in eukaryotes initiates at many origins distributed across each chromosome. Origins are bound by the origin recognition complex (ORC), which, with Cdc6 and Cdt1, recruits and loads the Mcm2-7 (MCM) helicase as an inactive double hexamer during G1 phase. The replisome assembles at the...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Nature Publishing Group UK
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998030/ https://www.ncbi.nlm.nih.gov/pubmed/33772005 http://dx.doi.org/10.1038/s41467-021-22216-x |
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author | Sánchez, Humberto McCluskey, Kaley van Laar, Theo van Veen, Edo Asscher, Filip M. Solano, Belén Diffley, John F. X. Dekker, Nynke H. |
author_facet | Sánchez, Humberto McCluskey, Kaley van Laar, Theo van Veen, Edo Asscher, Filip M. Solano, Belén Diffley, John F. X. Dekker, Nynke H. |
author_sort | Sánchez, Humberto |
collection | PubMed |
description | DNA replication in eukaryotes initiates at many origins distributed across each chromosome. Origins are bound by the origin recognition complex (ORC), which, with Cdc6 and Cdt1, recruits and loads the Mcm2-7 (MCM) helicase as an inactive double hexamer during G1 phase. The replisome assembles at the activated helicase in S phase. Although the outline of replisome assembly is understood, little is known about the dynamics of individual proteins on DNA and how these contribute to proper complex formation. Here we show, using single-molecule optical trapping and confocal microscopy, that yeast ORC is a mobile protein that diffuses rapidly along DNA. Origin recognition halts this search process. Recruitment of MCM molecules in an ORC- and Cdc6-dependent fashion results in slow-moving ORC-MCM intermediates and MCMs that rapidly scan the DNA. Following ATP hydrolysis, salt-stable loading of MCM single and double hexamers was seen, both of which exhibit salt-dependent mobility. Our results demonstrate that effective helicase loading relies on an interplay between protein diffusion and origin recognition, and suggest that MCM is stably loaded onto DNA in multiple forms. |
format | Online Article Text |
id | pubmed-7998030 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-79980302021-04-16 DNA replication origins retain mobile licensing proteins Sánchez, Humberto McCluskey, Kaley van Laar, Theo van Veen, Edo Asscher, Filip M. Solano, Belén Diffley, John F. X. Dekker, Nynke H. Nat Commun Article DNA replication in eukaryotes initiates at many origins distributed across each chromosome. Origins are bound by the origin recognition complex (ORC), which, with Cdc6 and Cdt1, recruits and loads the Mcm2-7 (MCM) helicase as an inactive double hexamer during G1 phase. The replisome assembles at the activated helicase in S phase. Although the outline of replisome assembly is understood, little is known about the dynamics of individual proteins on DNA and how these contribute to proper complex formation. Here we show, using single-molecule optical trapping and confocal microscopy, that yeast ORC is a mobile protein that diffuses rapidly along DNA. Origin recognition halts this search process. Recruitment of MCM molecules in an ORC- and Cdc6-dependent fashion results in slow-moving ORC-MCM intermediates and MCMs that rapidly scan the DNA. Following ATP hydrolysis, salt-stable loading of MCM single and double hexamers was seen, both of which exhibit salt-dependent mobility. Our results demonstrate that effective helicase loading relies on an interplay between protein diffusion and origin recognition, and suggest that MCM is stably loaded onto DNA in multiple forms. Nature Publishing Group UK 2021-03-26 /pmc/articles/PMC7998030/ /pubmed/33772005 http://dx.doi.org/10.1038/s41467-021-22216-x Text en © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Sánchez, Humberto McCluskey, Kaley van Laar, Theo van Veen, Edo Asscher, Filip M. Solano, Belén Diffley, John F. X. Dekker, Nynke H. DNA replication origins retain mobile licensing proteins |
title | DNA replication origins retain mobile licensing proteins |
title_full | DNA replication origins retain mobile licensing proteins |
title_fullStr | DNA replication origins retain mobile licensing proteins |
title_full_unstemmed | DNA replication origins retain mobile licensing proteins |
title_short | DNA replication origins retain mobile licensing proteins |
title_sort | dna replication origins retain mobile licensing proteins |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998030/ https://www.ncbi.nlm.nih.gov/pubmed/33772005 http://dx.doi.org/10.1038/s41467-021-22216-x |
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