Characterization of the dimeric CMG/pre-initiation complex and its transition into DNA replication forks

Area of Science:

• Molecular Biology
• Cellular Biology
• Biochemistry

Background:

• The pre-initiation complex (pre-IC) is a proposed intermediate in eukaryotic DNA replication fork maturation.
• Its existence and biochemical characteristics have remained largely unknown for two decades.

Purpose of the Study:

• To isolate and characterize the endogenous pre-IC.
• To provide in vivo evidence for the pre-IC/fork transition during replication initiation.

Main Methods:

• Utilized multiple enrichment strategies to isolate endogenous CMG-containing complexes.
• Analyzed complexes using mass spectrometry and electron microscopy.
• Assessed DNA unwinding and synthesis activities of isolated complexes.

Main Results:

• Isolated a novel dimeric CMG complex (d-CMG), distinct from single CMG (s-CMG).
• d-CMG is assembled during S phase and matures into s-CMG/replisome.
• RPA and DNA Pol α/primase associate with s-CMG, not d-CMG.
• Inactivation of DNA Pol α-primase increased d-CMG levels, indicating a stalled pre-IC/fork transition.

Conclusions:

• The study reveals the biochemical properties of d-CMG/pre-IC.
• Provides in vivo evidence supporting the pre-IC/fork transition as a critical step in replication initiation.
• Characterizes d-CMG as a distinct functional state preceding active replication.