Print Email Facebook Twitter Nucleotide binding halts diffusion of the eukaryotic replicative helicase during activation Title Nucleotide binding halts diffusion of the eukaryotic replicative helicase during activation Author Ramirez Montero, D.F. (TU Delft BN/Nynke Dekker Lab; Kavli institute of nanoscience Delft) Sanchez González, H. (TU Delft BN/Nynke Dekker Lab; Kavli institute of nanoscience Delft) van Veen, E.N.W. (TU Delft BN/Nynke Dekker Lab; TU Delft BN/Bionanoscience; Kavli institute of nanoscience Delft) van Laar, T. (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) Department BN/Bionanoscience Date 2023 Abstract The eukaryotic replicative helicase CMG centrally orchestrates the replisome and leads the way at the front of replication forks. Understanding the motion of CMG on the DNA is therefore key to our understanding of DNA replication. In vivo, CMG is assembled and activated through a cell-cycle-regulated mechanism involving 36 polypeptides that has been reconstituted from purified proteins in ensemble biochemical studies. Conversely, single-molecule studies of CMG motion have thus far relied on pre-formed CMG assembled through an unknown mechanism upon overexpression of individual constituents. Here, we report the activation of CMG fully reconstituted from purified yeast proteins and the quantification of its motion at the single-molecule level. We observe that CMG can move on DNA in two ways: by unidirectional translocation and by diffusion. We demonstrate that CMG preferentially exhibits unidirectional translocation in the presence of ATP, whereas it preferentially exhibits diffusive motion in the absence of ATP. We also demonstrate that nucleotide binding halts diffusive CMG independently of DNA melting. Taken together, our findings support a mechanism by which nucleotide binding allows newly assembled CMG to engage with the DNA within its central channel, halting its diffusion and facilitating the initial DNA melting required to initiate DNA replication. To reference this document use: http://resolver.tudelft.nl/uuid:deac6a5d-c41b-4642-86e8-f9c8317b011f DOI https://doi.org/10.1038/s41467-023-37093-9 ISSN 2041-1723 Source Nature Communications, 14 (1), 2082 Part of collection Institutional Repository Document type journal article Rights © 2023 D.F. Ramirez Montero, H. Sanchez González, E.N.W. van Veen, T. van Laar, B.P. Solano Hermosilla, John F.X. Diffley, N.H. Dekker Files PDF s41467_023_37093_9.pdf 1.96 MB Close viewer /islandora/object/uuid:deac6a5d-c41b-4642-86e8-f9c8317b011f/datastream/OBJ/view