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Sld3CBD-Cdc45 structural insights into Cdc45 recruitment for CMG complex formation during DNA replication
Hao Li1, Izumi Ishizaki1, Koji Kato1,2
1Graduate School of Life Science, Hokkaido University, Sapporo, Japan.
Elife
|September 8, 2025
Summary
The study reveals how Sld3 protein regulates the formation of the Cdc45-MCM-GINS (CMG) helicase complex during DNA replication. It shows Sld3 binding to Cdc45 and its dissociation upon unwound DNA, clarifying CMG complex regulation.
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- DNA replication initiation requires the assembly of the Cdc45-MCM-GINS (CMG) helicase at replication origins.
- The initiation factor Sld3, along with Sld7, plays a crucial role in recruiting Cdc45 and GINS to form the CMG complex.
- The precise mechanism by which Sld3 regulates CMG complex formation remains incompletely understood.
Purpose of the Study:
- To elucidate the structural basis and mechanism of Sld3-mediated regulation of CMG complex formation.
- To present the structure of the Sld3 Cdc45-binding domain (Sld3CBD) in complex with Cdc45.
- To investigate the interactions between Sld3, Cdc45, GINS, and MCM during CMG assembly.
Main Methods:
- X-ray crystallography to determine the structure of the Sld3CBD-Cdc45 complex.
- Mutant analysis to assess the importance of Sld3-Cdc45 interactions.
- Biochemical assays, including particle size analysis and DNA-binding assays.
- Modeling of the Sld3CBD-CMG complex and the Cdc45-MCM dimer.
Main Results:
- The crystal structure of Sld3CBD-Cdc45 reveals specific interactions and conformational changes essential for their binding.
- Mutant analysis confirmed the lability of the Sld3CBD-Cdc45 interaction.
- Structural modeling indicated that Sld3CBD, GINS, and MCM bind to distinct sites on Cdc45, allowing Sld7-Sld3 to remain associated with Cdc45-MCM until CMG formation.
- A DNA-binding assay demonstrated that Sld7-Sld3 dissociates from the CMG complex in the presence of unwound single-stranded DNA fragments.
Conclusions:
- Sld3 directly interacts with Cdc45 through its binding domain, influencing CMG complex assembly.
- The Sld7-Sld3 complex acts as a scaffold, remaining associated with Cdc45-MCM until CMG formation is complete.
- The dissociation of Sld7-Sld3 from CMG is linked to the presence of unwound single-stranded DNA, suggesting a regulatory role in replication initiation or progression.
- These findings provide critical insights into the molecular mechanisms governing CMG complex formation and regulation by Sld3.
Keywords:
DNA replication initiationE. coliK. marxianusS. cerevisiaebudding yeasteukaryotesinitiation factor Sld3molecular biophysicsrecruiting Cdc45structural biologyMore Related Videos
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