Structural insights into the Cdt1-mediated MCM2-7 chromatin loading.

Area of Science:

• Molecular Biology
• Cell Biology
• Structural Biology

Background:

• DNA replication initiation in eukaryotes is tightly regulated to occur once per cell cycle.
• The Cdt1 protein facilitates the loading of the mini-chromosome maintenance 2-7 (MCM2-7) helicase onto chromatin, a critical step for replication.
• Understanding the molecular mechanism of MCM2-7 loading is essential for comprehending cell division control.

Purpose of the Study:

• To determine the structure of the human Cdt1-Mcm6 binding domains complex.
• To elucidate the molecular mechanism of Cdt1-mediated MCM2-7 helicase loading onto chromatin.
• To investigate the role of charge complementarity in the Cdt1-Mcm2-7 interaction.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy was used to determine the structure of the Cdt1(410-440)/MCM6(708-821) complex.
• Site-directed mutagenesis was employed to study the functional significance of interacting residues.
• Yeast genetics was used to assess the impact of mutations on DNA replication and MCM2 loading.

Main Results:

• The structure revealed charge complementarity as a key determinant for the specific interaction between Cdt1 and Mcm2-7.
• Mutations disrupting this interaction in yeast Cdt1 and Mcm6 led to defects in DNA replication and MCM2 chromatin loading.
• These defects resulted in cell death, highlighting the critical nature of this interaction.
• The Cdt1-Mcm6 interaction occurs via their C-termini, suggesting a specific orientation of MCM2-7 during loading.

Conclusions:

• Charge complementarity is essential for the specific binding of Cdt1 to the MCM2-7 helicase.
• Disruption of the Cdt1-Mcm2-7 interaction impairs DNA replication initiation and leads to cell death.
• The findings provide a structural basis for Cdt1-mediated MCM2-7 loading, suggesting the MCM2-7 hexamer is loaded with its C-terminal end towards the ORC complex.