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Published on: July 26, 2024
MCM interference during licensing of DNA replication in Xenopus egg extracts-Possible Role of a C-terminal region of
Satoru Mimura1, Yumiko Kubota1, Haruhiko Takisawa1
1a Department of Biological Sciences, Graduate School of Science , Osaka University , Machikaneyama 1-1, Toyonaka , Osaka , Japan.
Abstract:
The minichromosome maintenance (MCM) complex, consisting of six subunits, Mcm2-7, is loaded onto replication origins through loading factors (origin recognition complex [ORC], Cdc6, and Cdt1) and forms an MCM double hexamer that licenses the initiation of DNA replication. Previous studies with Xenopus egg extracts showed that loading factors, especially Cdc6, dissociate from chromatin on MCM loading, but the molecular mechanism and physiological significance remain largely unknown. Using a cell-free system for MCM loading onto plasmid DNA in Xenopus egg extracts, we found that MCM loaded onto DNA prevents DNA binding of the loading factors ORC, Cdc6, and Cdt1. We further report that a peptide of the C-terminal region of MCM3 (MCM3-C), previously implicated in the initial association with ORC/Cdc6 in budding yeast, prevents ORC/Cdc6/Cdt1 binding to DNA in the absence of MCM loading. ATP-γ-S suppresses inhibitory activities of both the MCM loaded onto DNA and the MCM3-C peptide. Other soluble factors in the extract, but neither MCM nor Cdt1, are required for the activity. Conservation of the amino acid sequences of MCM3-C and its activity in vertebrates implies a novel negative autoregulatory mechanism that interferes with MCM loading in the vicinity of licensed origins to ensure proper origin licensing.
Insights
Minichromosome maintenance (MCM) complex loading onto DNA prevents further loading factor binding. A conserved MCM3 peptide acts as a negative regulator, ensuring proper DNA replication origin licensing.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The minichromosome maintenance (MCM) complex (Mcm2-7) is essential for DNA replication initiation.
- Loading factors (ORC, Cdc6, Cdt1) facilitate MCM complex loading onto replication origins.
- Previous studies suggested loading factors dissociate post-MCM loading, but mechanisms were unclear.
Purpose of the Study:
- To investigate the molecular mechanism of loading factor dissociation after MCM complex loading.
- To identify factors regulating MCM loading and origin licensing.
- To explore the role of MCM3 C-terminal region in regulating MCM loading.
Main Methods:
- Utilized a cell-free system with Xenopus egg extracts for MCM loading onto plasmid DNA.
- Investigated the effect of loaded MCM on DNA binding of loading factors (ORC, Cdc6, Cdt1).
- Examined the inhibitory activity of an MCM3 C-terminal peptide (MCM3-C) on loading factor DNA binding.
Main Results:
- MCM complex loaded onto DNA inhibits DNA binding of ORC, Cdc6, and Cdt1.
- The MCM3-C peptide prevents ORC/Cdc6/Cdt1 binding to DNA, independent of MCM loading.
- ATP-γ-S suppressed the inhibitory activities of both loaded MCM and MCM3-C.
- Soluble factors, excluding MCM and Cdt1, mediate this inhibitory activity.
Conclusions:
- Discovered a novel negative autoregulatory mechanism for MCM loading.
- MCM complex and MCM3-C peptide interfere with loading factor binding near licensed origins.
- This mechanism ensures precise origin licensing for proper DNA replication.
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