The Dynamics of Eukaryotic Replication Initiation: Origin Specificity, Licensing, and Firing at the Single-Molecule Level
Eukaryotic replication initiation is highly regulated and dynamic. It begins with the origin recognition complex (ORC) binding DNA sites called origins of replication. ORC, together with Cdc6 and Cdt1, mediate pre-replicative complex (pre-RC) assembly by loading a double hexamer of Mcm2–7: the core...
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| Format: | Article |
| Language: | en_US |
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Elsevier
2017
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| Online Access: | http://hdl.handle.net/1721.1/106210 https://orcid.org/0000-0003-1736-6343 https://orcid.org/0000-0002-0124-0087 https://orcid.org/0000-0002-2876-610X |
| _version_ | 1826215517862494208 |
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| author | Duzdevich, Daniel Ticau, Simina Greene, Eric C. Warner, Megan Dobbins Ivica, Nikola Bell, Stephen P |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Duzdevich, Daniel Ticau, Simina Greene, Eric C. Warner, Megan Dobbins Ivica, Nikola Bell, Stephen P |
| author_sort | Duzdevich, Daniel |
| collection | MIT |
| description | Eukaryotic replication initiation is highly regulated and dynamic. It begins with the origin recognition complex (ORC) binding DNA sites called origins of replication. ORC, together with Cdc6 and Cdt1, mediate pre-replicative complex (pre-RC) assembly by loading a double hexamer of Mcm2–7: the core of the replicative helicase. Here, we use single-molecule imaging to directly visualize Saccharomyces cerevisiae pre-RC assembly and replisome firing in real time. We show that ORC can locate and stably bind origins within large tracts of non-origin DNA and that Cdc6 drives ordered pre-RC assembly. We further show that the dynamics of the ORC-Cdc6 interaction dictate Mcm2–7 loading specificity and that Mcm2–7 double hexamers form preferentially at a native origin sequence. Finally, we demonstrate that single Mcm2–7 hexamers propagate bidirectionally, monotonically, and processively as constituents of active replisomes. |
| first_indexed | 2024-09-23T16:32:53Z |
| format | Article |
| id | mit-1721.1/106210 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T16:32:53Z |
| publishDate | 2017 |
| publisher | Elsevier |
| record_format | dspace |
| spelling | mit-1721.1/1062102022-10-02T08:17:26Z The Dynamics of Eukaryotic Replication Initiation: Origin Specificity, Licensing, and Firing at the Single-Molecule Level Duzdevich, Daniel Ticau, Simina Greene, Eric C. Warner, Megan Dobbins Ivica, Nikola Bell, Stephen P Massachusetts Institute of Technology. Department of Biology Warner, Megan Dobbins Ivica, Nikola Bell, Stephen P Eukaryotic replication initiation is highly regulated and dynamic. It begins with the origin recognition complex (ORC) binding DNA sites called origins of replication. ORC, together with Cdc6 and Cdt1, mediate pre-replicative complex (pre-RC) assembly by loading a double hexamer of Mcm2–7: the core of the replicative helicase. Here, we use single-molecule imaging to directly visualize Saccharomyces cerevisiae pre-RC assembly and replisome firing in real time. We show that ORC can locate and stably bind origins within large tracts of non-origin DNA and that Cdc6 drives ordered pre-RC assembly. We further show that the dynamics of the ORC-Cdc6 interaction dictate Mcm2–7 loading specificity and that Mcm2–7 double hexamers form preferentially at a native origin sequence. Finally, we demonstrate that single Mcm2–7 hexamers propagate bidirectionally, monotonically, and processively as constituents of active replisomes. National Institutes of Health (U.S.) (Grant GM52339) National Institutes of Health (U.S.) (Pre-Doctoral Training Grant GM007287) National Science Foundation (U.S.) (Graduate Research Fellowship 1122374) 2017-01-05T19:24:21Z 2017-01-05T19:24:21Z 2015-05 Article http://purl.org/eprint/type/JournalArticle 10972765 http://hdl.handle.net/1721.1/106210 Duzdevich, Daniel et al. “The Dynamics of Eukaryotic Replication Initiation: Origin Specificity, Licensing, and Firing at the Single-Molecule Level.” Molecular Cell 58.3 (2015): 483–494. https://orcid.org/0000-0003-1736-6343 https://orcid.org/0000-0002-0124-0087 https://orcid.org/0000-0002-2876-610X en_US http://dx.doi.org/10.1016/j.molcel.2015.03.017 Molecular Cell Creative Commons Attribution-NonCommercial-NoDerivs License http://creativecommons.org/licenses/by-nc-nd/4.0/ application/pdf Elsevier PMC |
| spellingShingle | Duzdevich, Daniel Ticau, Simina Greene, Eric C. Warner, Megan Dobbins Ivica, Nikola Bell, Stephen P The Dynamics of Eukaryotic Replication Initiation: Origin Specificity, Licensing, and Firing at the Single-Molecule Level |
| title | The Dynamics of Eukaryotic Replication Initiation: Origin Specificity, Licensing, and Firing at the Single-Molecule Level |
| title_full | The Dynamics of Eukaryotic Replication Initiation: Origin Specificity, Licensing, and Firing at the Single-Molecule Level |
| title_fullStr | The Dynamics of Eukaryotic Replication Initiation: Origin Specificity, Licensing, and Firing at the Single-Molecule Level |
| title_full_unstemmed | The Dynamics of Eukaryotic Replication Initiation: Origin Specificity, Licensing, and Firing at the Single-Molecule Level |
| title_short | The Dynamics of Eukaryotic Replication Initiation: Origin Specificity, Licensing, and Firing at the Single-Molecule Level |
| title_sort | dynamics of eukaryotic replication initiation origin specificity licensing and firing at the single molecule level |
| url | http://hdl.handle.net/1721.1/106210 https://orcid.org/0000-0003-1736-6343 https://orcid.org/0000-0002-0124-0087 https://orcid.org/0000-0002-2876-610X |
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