DNA replication origins retain mobile licensing proteins
Autor: | Humberto Sanchez, Edo van Veen, Nynke H. Dekker, Kaley McCluskey, John F.X. Diffley, Belen Solano, Theo van Laar, Filip M. Asscher |
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Rok vydání: | 2021 |
Předmět: |
DNA Replication
0301 basic medicine Saccharomyces cerevisiae Proteins Science Origin Recognition Complex General Physics and Astronomy Cell Cycle Proteins Replication Origin Saccharomyces cerevisiae Random hexamer Article General Biochemistry Genetics and Molecular Biology DNA replication factor CDT1 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Single-molecule biophysics DNA Fungal Binding Sites Multidisciplinary Minichromosome Maintenance Proteins Models Genetic biology Chemistry DNA replication Fungal genetics Helicase General Chemistry Cell biology DNA-Binding Proteins 030104 developmental biology biology.protein Replisome Origin recognition complex Origin selection Algorithms 030217 neurology & neurosurgery DNA Protein Binding |
Zdroj: | Nature Communications, Vol 12, Iss 1, Pp 1-12 (2021) Nature Communications, 12(1) Nature Communications |
ISSN: | 2041-1723 |
DOI: | 10.1038/s41467-021-22216-x |
Popis: | 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. Eukaryotic DNA replication is regulated to ensure copying of the genome (only) once per cell cycle. Here the authors, using optical trapping and confocal microscopy, demonstrate the dynamics of the origin recognition complex and subsequent intermediates that lead up to the loading of an MCM helicase onto DNA. |
Databáze: | OpenAIRE |
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