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Colocalization of MCM8 and MCM7 with proteins involved in distinct aspects of DNA replication
Yayoi Kinoshita1, Edward M Johnson, Ronald E Gordon
1Department of Pathology, Mount Sinai School of Medicine, New York, New York 10029, USA.
Abstract:
Minichromosome maintenance (MCM) proteins are essential for DNA replication in eukaryotes. A subcomplex of the MCM2-7 family members, initially characterized in yeast, is thought to serve as a eukaryotic DNA replicative helicase. MCM8 is a new family member, not present in yeast, which may function alone or with other family members in aspects of DNA metabolism, including replication initiation and elongation. Through the use of chromatin immunoprecipitation, we find that MCM8, like MCM7, colocalizes on a specific DNA segment of the c-MYC replication initiation zone (c-MYC replicator) with Cdc6, a protein potentially involved in loading MCM proteins onto DNA. The association between MCM8 and MCM7 peaks in mid G1, at the time of assembly of the prereplication complex. The association of both MCM proteins with Cdc6, however, continues even after DNA replication is complete. We also find that MCM8 colocalizes at the c-MYC replicator with chromatin-bound Cdk2. Our data indicate that any role MCM8 may play in elongation is likely to be discontinuous, in its association with DNA, from a potential role in initiation. Using immunogold electron microscopy we show that MCM8 and MCM7 differ in spatial relation to RPA70 during S phase. Our data strongly suggest that MCM8 functions with other known replication proteins in processes which accompany DNA replication, especially initiation, and which are specifically adapted to suit higher eukaryotes.
Insights
Minichromosome maintenance 8 (MCM8) protein associates with DNA replication initiation sites, particularly the c-MYC replicator, and interacts with key replication proteins like Cdc6 and Cdk2 in higher eukaryotes.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Minichromosome maintenance (MCM) proteins form the core of the eukaryotic DNA replicative helicase.
- MCM8 is a recently identified MCM family member, absent in yeast, potentially involved in DNA replication.
- Understanding MCM8's function is crucial for comprehending DNA replication regulation in higher eukaryotes.
Purpose of the Study:
- To investigate the role and localization of the MCM8 protein during DNA replication.
- To determine MCM8's association with other replication factors and its presence at replication origins.
- To elucidate MCM8's specific function in DNA replication initiation and elongation in higher eukaryotes.
Main Methods:
- Chromatin immunoprecipitation (ChIP) to identify MCM8 binding sites on DNA.
- Analysis of MCM8 and MCM7 colocalization with Cdc6 and Cdk2 at the c-MYC replicator.
- Immunogold electron microscopy to examine the spatial relationship between MCM8, MCM7, and RPA70.
Main Results:
- MCM8 colocalizes with MCM7 and Cdc6 at the c-MYC replication initiation zone (replicator), peaking during G1 phase.
- MCM8 also associates with chromatin-bound Cdk2 at the c-MYC replicator.
- Distinct spatial relationships between MCM8, MCM7, and RPA70 were observed during S phase, suggesting differential roles.
Conclusions:
- MCM8 plays a significant role in DNA replication initiation, particularly at specific origins like the c-MYC replicator.
- Its association with replication proteins and distinct localization suggest a specialized function in higher eukaryotic DNA replication.
- MCM8's function in elongation appears discontinuous and separate from its role in initiation.
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