Mehanna A and Diffley JF (2012) Pre-replicative complex assembly with purified proteins. Methods 57(2):222-6
Abstract: Licensing of origins of eukaryotic DNA replication involves the loading of six minichromosome maintenance proteins (Mcm2-7) into pre-replicative complexes (pre-RCs). The assembly of the pre-RC is restricted to G1 phase of the cell cycle, which is crucial to ensure once per cell cycle DNA replication. Mcm2-7 is loaded by the action of the origin recognition complex (ORC), Cdc6 and Cdt1 and requires ATP. In vitro reconstitution of this reaction has shown that Mcm2-7 is loaded onto DNA as a symmetrical head-to-head double hexamer. We describe in detail how pre-RC proteins are purified and used to reconstitute pre-RC formation in vitro. This method is useful for studying the biochemical mechanisms of Mcm2-7 loading as well as subsequent steps in DNA replication.
|Status: Published||Type: Journal Article | Research Support, Non-U.S. Gov't||PubMed ID: 22732456|
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