Print Email Facebook Twitter DNA replication origins retain mobile licensing proteins Title DNA replication origins retain mobile licensing proteins Author Sanchez González, H. (TU Delft BN/Nynke Dekker Lab; Kavli institute of nanoscience Delft) Mc Cluskey, K.A. (TU Delft BN/Nynke Dekker Lab; Kavli institute of nanoscience Delft) van Laar, T. (TU Delft BN/Nynke Dekker Lab; Kavli institute of nanoscience Delft) van Veen, E.N.W. (TU Delft BN/Nynke Dekker Lab; Kavli institute of nanoscience Delft) Asscher, F.M. (TU Delft BN/Nynke Dekker Lab; Kavli institute of nanoscience Delft) Solano Hermosilla, B.P. (TU Delft BN/Nynke Dekker Lab; Kavli institute of nanoscience Delft) Diffley, John F.X. (Francis Crick Institute) Dekker, N.H. (TU Delft BN/Nynke Dekker Lab; Kavli institute of nanoscience Delft) Date 2021 Abstract 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. To reference this document use: http://resolver.tudelft.nl/uuid:9dbccabe-6a49-4879-beb7-c77bfdc96524 DOI https://doi.org/10.1038/s41467-021-22216-x ISSN 2041-1723 Source Nature Communications, 12 (1) Part of collection Institutional Repository Document type journal article Rights © 2021 H. Sanchez González, K.A. Mc Cluskey, T. van Laar, E.N.W. van Veen, F.M. Asscher, B.P. Solano Hermosilla, John F.X. Diffley, N.H. Dekker Files PDF s41467_021_22216_x.pdf 3.68 MB Close viewer /islandora/object/uuid:9dbccabe-6a49-4879-beb7-c77bfdc96524/datastream/OBJ/view