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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
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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.

Keywords:
DNA replication initiationE. coliK. marxianusS. cerevisiaebudding yeasteukaryotesinitiation factor Sld3molecular biophysicsrecruiting Cdc45structural biology

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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.