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.
Clare Hall Laboratories, Cancer Research UK, South Mimms, UK.
Amina
A
Mehanna
John F X
JF
Diffley
D002021:Buffers; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002868:Chromosomal Proteins, Non-Histone / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D004261:DNA Replication; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D051738:Origin Recognition Complex / Q000737:chemistry / Q000302:isolation & purification; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D018741:Replication Origin; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000302:isolation & purification
D002021:Buffers; C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004271:DNA, Fungal; D051738:Origin Recognition Complex; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins