Nucleotide binding halts diffusion of the eukaryotic replicative helicase during activation
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...
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| Format: | Article |
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Nature Portfolio
2023-04-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-023-37093-9 |
| _version_ | 1797845902180745216 |
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| author | Daniel Ramírez Montero Humberto Sánchez Edo van Veen Theo van Laar Belén Solano John F. X. Diffley Nynke H. Dekker |
| author_facet | Daniel Ramírez Montero Humberto Sánchez Edo van Veen Theo van Laar Belén Solano John F. X. Diffley Nynke H. Dekker |
| author_sort | Daniel Ramírez Montero |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-09T17:46:28Z |
| format | Article |
| id | doaj.art-ccb1945bc66942e4ba899cf6a738ad2d |
| institution | Directory Open Access Journal |
| issn | 2041-1723 |
| language | English |
| last_indexed | 2024-04-09T17:46:28Z |
| publishDate | 2023-04-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj.art-ccb1945bc66942e4ba899cf6a738ad2d2023-04-16T11:19:44ZengNature PortfolioNature Communications2041-17232023-04-0114111510.1038/s41467-023-37093-9Nucleotide binding halts diffusion of the eukaryotic replicative helicase during activationDaniel Ramírez Montero0Humberto Sánchez1Edo van Veen2Theo van Laar3Belén Solano4John F. X. Diffley5Nynke H. Dekker6Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of TechnologyDepartment of Bionanoscience, Kavli Institute of Nanoscience, Delft University of TechnologyDepartment of Bionanoscience, Kavli Institute of Nanoscience, Delft University of TechnologyDepartment of Bionanoscience, Kavli Institute of Nanoscience, Delft University of TechnologyDepartment of Bionanoscience, Kavli Institute of Nanoscience, Delft University of TechnologyChromosome Replication Laboratory, Francis Crick InstituteDepartment of Bionanoscience, Kavli Institute of Nanoscience, Delft University of TechnologyAbstract 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.https://doi.org/10.1038/s41467-023-37093-9 |
| spellingShingle | Daniel Ramírez Montero Humberto Sánchez Edo van Veen Theo van Laar Belén Solano John F. X. Diffley Nynke H. Dekker Nucleotide binding halts diffusion of the eukaryotic replicative helicase during activation Nature Communications |
| title | Nucleotide binding halts diffusion of the eukaryotic replicative helicase during activation |
| title_full | Nucleotide binding halts diffusion of the eukaryotic replicative helicase during activation |
| title_fullStr | Nucleotide binding halts diffusion of the eukaryotic replicative helicase during activation |
| title_full_unstemmed | Nucleotide binding halts diffusion of the eukaryotic replicative helicase during activation |
| title_short | Nucleotide binding halts diffusion of the eukaryotic replicative helicase during activation |
| title_sort | nucleotide binding halts diffusion of the eukaryotic replicative helicase during activation |
| url | https://doi.org/10.1038/s41467-023-37093-9 |
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