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Updated: Apr 5, 2026
Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method
Published on: May 2, 2025
Conserved mechanism for coordinating replication fork helicase assembly with phosphorylation of the helicase
Irina Bruck1, Daniel L Kaplan2
1Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL 32306.
Abstract:
Dbf4-dependent kinase (DDK) phosphorylates minichromosome maintenance 2 (Mcm2) during S phase in yeast, and Sld3 recruits cell division cycle 45 (Cdc45) to minichromosome maintenance 2-7 (Mcm2-7). We show here DDK-phosphoryled Mcm2 preferentially interacts with Cdc45 in vivo, and that Sld3 stimulates DDK phosphorylation of Mcm2 by 11-fold. We identified a mutation of the replication initiation factor Sld3, Sld3-m16, that is specifically defective in stimulating DDK phosphorylation of Mcm2. Wild-type expression levels of sld3-m16 result in severe growth and DNA replication defects. Cells expressing sld3-m16 exhibit no detectable Mcm2 phosphorylation in vivo, reduced replication protein A-ChIP signal at an origin, and diminished Go, Ichi, Ni, and San association with Mcm2-7. Treslin, the human homolog of Sld3, stimulates human DDK phosphorylation of human Mcm2 by 15-fold. DDK phosphorylation of human Mcm2 decreases the affinity of Mcm5 for Mcm2, suggesting a potential mechanism for helicase ring opening. These data suggest a conserved mechanism for replication initiation: Sld3/Treslin coordinates Cdc45 recruitment to Mcm2-7 with DDK phosphorylation of Mcm2 during S phase.
Insights
Sld3 protein stimulates Dbf4-dependent kinase (DDK) phosphorylation of Mcm2, a crucial step in DNA replication initiation. This conserved mechanism involves Sld3/Treslin coordinating Cdc45 recruitment and Mcm2 phosphorylation during S phase.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Dbf4-dependent kinase (DDK) phosphorylates minichromosome maintenance 2 (Mcm2) during S phase in yeast.
- Sld3 protein is known to recruit cell division cycle 45 (Cdc45) to the Mcm2-7 complex.
Purpose of the Study:
- To investigate the interaction between DDK-phosphorylated Mcm2 and Cdc45.
- To elucidate the role of Sld3 in stimulating DDK phosphorylation of Mcm2.
- To explore the conserved mechanism of DNA replication initiation in yeast and humans.
Main Methods:
- In vivo interaction studies.
- Site-directed mutagenesis to identify Sld3 mutations affecting DDK phosphorylation.
- Cell growth and DNA replication defect analysis.
- Chromatin immunoprecipitation (ChIP) assays.
- Analysis of protein-Mcm2-7 complex association.
Main Results:
- DDK-phosphorylated Mcm2 preferentially interacts with Cdc45 in vivo.
- Sld3 significantly stimulates DDK phosphorylation of Mcm2 (11-fold in yeast, 15-fold in human cells via Treslin).
- A specific Sld3 mutation (Sld3-m16) impairs its ability to stimulate DDK phosphorylation, leading to severe growth and DNA replication defects, and reduced Mcm2 phosphorylation.
- The human homolog of Sld3, Treslin, also stimulates DDK phosphorylation of human Mcm2.
- DDK phosphorylation of human Mcm2 reduces Mcm5 binding affinity, suggesting a role in helicase ring opening.
Conclusions:
- Sld3/Treslin plays a conserved role in DNA replication initiation by coordinating Cdc45 recruitment to Mcm2-7 with DDK-mediated Mcm2 phosphorylation.
- This process is essential for proper S phase progression and cell viability.
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