Pre-replicative complex assembly with purified proteins.
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.
Mesh Terms:
Buffers, Cell Cycle Proteins, Chromosomal Proteins, Non-Histone, DNA Replication, DNA, Fungal, Origin Recognition Complex, Protein Multimerization, Recombinant Proteins, Replication Origin, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins
Buffers, Cell Cycle Proteins, Chromosomal Proteins, Non-Histone, DNA Replication, DNA, Fungal, Origin Recognition Complex, Protein Multimerization, Recombinant Proteins, Replication Origin, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins
Methods
Date: Jun. 01, 2012
PubMed ID: 22732456
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