Mouse pre-replicative complex proteins colocalise and interact with the centrosome.

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• Eukaryotic DNA replication initiation involves sequential protein binding to origins.
• The Origin Recognition Complex (ORC) and Minichromosome Maintenance (MCM) complexes are key players in DNA replication.
• The role of these complexes beyond replication initiation, particularly in mitosis, is less understood.

Purpose of the Study:

• To investigate the localization and function of ORC and MCM proteins during mitosis.
• To explore the interaction of ORC and MCM proteins with mitotic regulators like polo-like kinase 1 (Plk1).
• To determine the consequences of disrupting ORC and MCM function on cell division.

Main Methods:

• Immunofluorescence microscopy using EGFP-tagged proteins.
• Yeast two-hybrid assays to study protein-protein interactions.
• Co-immunoprecipitation experiments.
• In vitro phosphorylation assays.
• Small interfering RNA (siRNA) for gene silencing.

Main Results:

• ORC and MCM subunits localize to centrosomes and co-localize with Plk1.
• Plk1 interacts with Mcm2 and Orc2.
• Gamma-tubulin interacts with Mcm3 and Orc2.
• Orc2 is a phosphorylation substrate of Plk1.
• Depletion of Orc2 and Mcm3 inhibits cell proliferation, alters cell cycle distribution, and leads to multinucleated cells with disorganized microtubules.

Conclusions:

• MCM and ORC proteins have significant roles in mitosis, extending beyond their established function in pre-replicative complex formation.
• These proteins interact with key mitotic kinase Plk1 and are involved in proper cell division and microtubule organization.
• Disruption of ORC and MCM function during mitosis has severe consequences for cell proliferation and integrity.