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Cdt1 forms a complex with the minichromosome maintenance protein (MCM) and activates its helicase activity

Zhiying You1, Hisao Masai

  • 1Genome Dynamics Project, Tokyo Metropolitan Institute of Medical Science, 18-22 Honkomagome 3-chome, Bunkyo-ku, Tokyo 113-8613, Japan.

The Journal of Biological Chemistry
|July 9, 2008
PubMed

Insights

Cdt1 protein is essential for efficient DNA replication by stimulating the DNA binding and helicase activities of the Mcm4/6/7 complex, crucial for pre-replication complex formation.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cell Biology

Background:

  • The Mcm4/6/7 complex exhibits DNA helicase activity, indicating its central role in DNA replication.
  • Cdt1 is known to be essential for loading Mcm onto chromatin, but its specific function in pre-replication complex (pre-RC) formation and replication initiation remains unclear.

Purpose of the Study:

  • To elucidate the precise role of Cdt1 in the function of the Mcm4/6/7 complex during DNA replication.
  • To investigate the interaction between Cdt1 and Mcm proteins and its impact on helicase activity.

Main Methods:

  • Glycerol gradient fractionation using purified proteins to analyze complex formation.
  • In vitro assays to assess DNA binding and helicase activities.
  • Site-directed mutagenesis of Cdt1 to study the functional consequences of impaired Mcm interaction.

Main Results:

  • Cdt1 forms complexes with various Mcm forms (Mcm4/6/7, Mcm2/3/4/5/6/7, Mcm2/4/6/7) through interaction with Mcm2 and Mcm4/6.
  • Cdt1 binding to Mcm4/6/7, particularly in the presence of ATP, forms a larger complex that significantly stimulates DNA binding and helicase activities.
  • A Cdt1 mutant deficient in Mcm interaction and high molecular weight complex formation failed to stimulate Mcm4/6/7 activity.

Conclusions:

  • A productive interaction between Cdt1 and the MCM complex is critical for efficient loading of MCM onto DNA.
  • Cdt1's stimulation of Mcm4/6/7 DNA binding and helicase activity is essential for efficient DNA unwinding during replication initiation.

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