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Methods. 2012 Jun;57(2):222-6. doi: 10.1016/j.ymeth.2012.06.008. Epub 2012 Jun 23.

Pre-replicative complex assembly with purified proteins.

Author information

1
Clare Hall Laboratories, Cancer Research UK, South Mimms, UK.

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.

PMID:
22732456
DOI:
10.1016/j.ymeth.2012.06.008
[Indexed for MEDLINE]

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