Prereplicative complexes assembled in vitro support origin-dependent and independent DNA replication.
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Embargo End Date
ICR Authors
Authors
On, KF
Beuron, F
Frith, D
Snijders, AP
Morris, EP
Diffley, JFX
Beuron, F
Frith, D
Snijders, AP
Morris, EP
Diffley, JFX
Document Type
Journal Article
Date
2014-03-18
Date Accepted
Abstract
Eukaryotic DNA replication initiates from multiple replication origins. To ensure each origin fires just once per cell cycle, initiation is divided into two biochemically discrete steps: the Mcm2-7 helicase is first loaded into prereplicative complexes (pre-RCs) as an inactive double hexamer by the origin recognition complex (ORC), Cdt1 and Cdc6; the helicase is then activated by a set of "firing factors." Here, we show that plasmids containing pre-RCs assembled with purified proteins support complete and semi-conservative replication in extracts from budding yeast cells overexpressing firing factors. Replication requires cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK). DDK phosphorylation of Mcm2-7 does not by itself promote separation of the double hexamer, but is required for the recruitment of firing factors and replisome components in the extract. Plasmid replication does not require a functional replication origin; however, in the presence of competitor DNA and limiting ORC concentrations, replication becomes origin-dependent in this system. These experiments indicate that Mcm2-7 double hexamers can be precursors of replication and provide insight into the nature of eukaryotic DNA replication origins.
Citation
The EMBO journal, 2014, 33 (6), pp. 605 - 620
Source Title
Publisher
WILEY-BLACKWELL
ISSN
0261-4189
eISSN
1460-2075
Collections
Research Team
Structural Electron Microscopy