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MCM3 complex required for cell cycle regulation of DNA replication in vertebrate cells
M A Madine1, C Y Khoo, A D Mills
1Wellcome/CRC Institute, Cambridge, UK.
Abstract:
An intact nuclear membrane restricts DNA replication to only one round in each cell cycle, apparently by excluding an essential replication-licensing factor throughout interphase. A family of related yeast replication proteins, MCM2, 3 and 5 (also called, after cell-division cycle, CDC46), resemble licensing factor, entering the nucleus only during mitosis. We have cloned a Xenopus homologue of MCM3 (XMCM3) and raised antibodies against expressed protein. Immunodepletion of Xenopus egg extracts removes a complex of MCM2, 3 and 5 homologues and inhibits replication of Xenopus sperm nuclei or permeable G2 HeLa nuclei. However, G1 HeLa nuclei still replicate efficiently. Mock-depleted extracts replicate all three templates. XMCM3 accumulates in nuclei before replication but anti-XMCM3 staining decreases during replication. These results can explain why replicated nuclei are unable to reinitiate replication in a single cell cycle.
Insights
A key replication-licensing factor, Xenopus MCM3 (XMCM3), enters the nucleus during mitosis but is excluded during interphase. Depletion of XMCM3 inhibits DNA replication, explaining the cell cycle
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The nuclear membrane regulates DNA replication, permitting only one round per cell cycle.
- This regulation is mediated by a replication-licensing factor excluded from the nucleus during interphase.
Purpose of the Study:
- To investigate the role of Xenopus MCM3 (XMCM3) in DNA replication licensing.
- To determine how XMCM3's nuclear localization influences replication control.
Main Methods:
- Cloning of Xenopus MCM3 (XMCM3) and antibody generation.
- Immunodepletion of Xenopus egg extracts to remove MCM protein complexes.
- Assessing DNA replication efficiency using Xenopus sperm nuclei and G1/G2 HeLa nuclei.
Main Results:
- Depletion of XMCM3 and associated proteins inhibited replication of sperm and G2 nuclei, but not G1 nuclei.
- XMCM3 accumulated in nuclei prior to replication and decreased during replication.
- Mock-depleted extracts supported replication of all tested nuclear types.
Conclusions:
- XMCM3 is essential for DNA replication licensing in Xenopus.
- The dynamic nuclear localization of XMCM3 is critical for preventing re-replication within a single cell cycle.