Prereplicative complexes assembled in vitro support origin-dependent and independent DNA replication

• Kin Fan On ^[1] ; Fabienne Beuron ^[2] ; David Frith ^[1] ; Ambrosius P Snijders ^[1] ; Edward P Morris ^[2] ; John F X Diffley ^[1]
  1. [1] London Research Institute

     London Research Institute

     Reino Unido

     
  2. [2] Institute of Cancer Research

     Institute of Cancer Research

     Reino Unido

     
• Localización: EMBO journal: European Molecular Biology Organization, ISSN 0261-4189, Vol. 33, Nº. 6, 2014, págs. 605-620
• Idioma: inglés
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• Resumen

  • 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.