Multiple Cdt1 molecules act at each origin to load replication-competent Mcm2-7 helicases.

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• DNA replication initiation in eukaryotes requires the Mcm2-7 replicative helicase to be loaded as a double hexamer at replication origins.
• Cdt1 protein plays a critical but transient role in this helicase loading process, though its precise mechanism remains unclear.

Purpose of the Study:

• To elucidate the mechanism by which Cdt1 facilitates Mcm2-7 helicase loading at eukaryotic origins of replication.
• To investigate the distinct functional domains of Cdt1 and their roles in origin recruitment and helicase loading.

Main Methods:

• Analysis of Cdt1 mutations to dissect its functions during helicase loading.
• Biochemical assays to study the interactions between Cdt1, Mcm2-7, ORC, and Cdc6.
• Investigating the role of Cdt1's N-terminal and C-terminal domains in Mcm2-7 loading and replication competence.

Main Results:

• The C-terminus of Cdt1 is essential for binding Mcm2-7 and recruiting it to origins.
• Origin recognition complex (ORC) and Cdc6 recruit multiple Cdt1 molecules, forming an intermediate essential for helicase loading.
• The N-terminal domain of Cdt1 is required for loading Mcm2-7 complexes competent for replication initiation.

Conclusions:

• Cdt1 acts through multiple functional domains to ensure efficient and correct loading of the Mcm2-7 helicase.
• A model is proposed where ORC and Cdc6 recruit two Cdt1 molecules to facilitate double-hexamer formation and subsequent Mcm2-7 loading.
• Cdt1's function extends beyond mere loading to influencing the replication competence of the Mcm2-7 helicase.