Prereplicative complexes assembled in vitro support origin-dependent and independent DNA replication.
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.
Collections
Subject
Saccharomycetales
Multiprotein Complexes
Protein-Serine-Threonine Kinases
Cell Cycle Proteins
Saccharomyces cerevisiae Proteins
DNA Replication
Enzyme Activation
Phosphorylation
Replication Origin
Plasmids
Models, Biological
Models, Molecular
Mass Spectrometry
Minichromosome Maintenance Proteins
Research team
Structural Electron Microscopy
Language
eng
License start date
2014-03
Citation
The EMBO journal, 2014, 33 (6), pp. 605 - 620
Publisher
WILEY-BLACKWELL