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Published on: December 14, 2015
MCM2-7 form double hexamers at licensed origins in Xenopus egg extract
Agnieszka Gambus1, Guennadi A Khoudoli, Richard C Jones
1Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, United Kingdom.
Abstract:
In late mitosis and G1, Mcm2-7 are assembled onto replication origins to license them for initiation in the upcoming S phase. After initiation, Mcm2-7 provide helicase activity to unwind DNA at the replication fork. Here we examine the structure of Mcm2-7 on chromatin in Xenopus egg extracts. We show that prior to replication initiation, Mcm2-7 is present at licensed replication origins in a complex with a molecular mass close to double that of the Mcm2-7 hexamer. This complex has approximately stoichiometric quantities of the 6 Mcm2-7 proteins and we conclude that it consists of a double heterohexamer. This provides a configuration potentially capable of initiating a pair of bidirectional replication forks in S phase. We also show that after initiation, Mcm2-7 associate with Cdc45 and GINS to form a relatively stable CMG (Cdc45-MCM-GINS) complex. The CMG proteins also associate less strongly with other replication proteins, consistent with the idea that a single CMG complex forms the core of the replisome.
Insights
Before DNA replication, Mcm2-7 proteins form a double hexamer at origins. After initiation, Mcm2-7, Cdc45, and GINS form the CMG complex, the core of the replisome.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Mcm2-7 proteins are essential for DNA replication licensing and unwinding.
- Understanding the structure and composition of Mcm2-7 complexes is crucial for comprehending replication initiation.
Purpose of the Study:
- To investigate the structural organization of Mcm2-7 complexes on chromatin before and after DNA replication initiation.
- To elucidate the composition of the CMG (Cdc45-MCM-GINS) complex and its role in the replisome.
Main Methods:
- Analysis of Mcm2-7 complexes in Xenopus egg extracts using biochemical assays.
- Determination of molecular mass and stoichiometry of protein complexes.
Main Results:
- Prior to initiation, Mcm2-7 forms a double heterohexamer complex at licensed replication origins.
- After initiation, Mcm2-7 associates with Cdc45 and GINS to form a stable CMG complex.
- The CMG complex interacts with other replication proteins, suggesting it forms the replisome core.
Conclusions:
- The double heterohexameric Mcm2-7 configuration is poised for initiating bidirectional replication forks.
- The CMG complex is a stable entity central to the DNA replication machinery.
