28. In eukaryotic replication, helicase loading occurs at all replicators during

(1) G_o phase            (2) G₁ phase

(3) S phase             (4) G₂ phase

Introduction

DNA replication in eukaryotic cells is a highly regulated process that ensures the genome is duplicated once and only once per cell cycle. A critical early step in this process is the loading of the replicative helicase onto DNA replication origins. This event, known as helicase loading, occurs exclusively during the G1 phase of the cell cycle. Understanding why helicase loading is confined to G1 and how it is regulated is essential for grasping the fidelity and timing of eukaryotic DNA replication.

What Is Helicase Loading?

Helicase loading refers to the assembly of the replicative DNA helicase complex, primarily the Mcm2-7 complex, onto replication origins. The helicase is loaded as an inactive double hexamer encircling double-stranded DNA (dsDNA). This loaded helicase forms part of the pre-replicative complex (pre-RC), which licenses the origin for replication in the subsequent S phase.

Why Does Helicase Loading Occur in G1 Phase?

1. Temporal Separation to Prevent Re-Replication

   • Helicase loading is restricted to G1 phase to ensure that each origin fires only once per cell cycle.

   • After G1, during S phase, the loaded helicase is activated to unwind DNA and initiate replication.

   • Preventing helicase loading outside G1 avoids re-licensing and re-replication of DNA, which can cause genome instability.

2. Pre-RC Formation in G1

   • The Origin Recognition Complex (ORC), along with accessory factors Cdc6 and Cdt1, recruits and loads Mcm2-7 helicases during G1.

   • This assembly creates a licensed origin ready for activation in S phase.

3. Regulatory Controls

   • Cyclin-dependent kinases (CDKs) and other regulatory proteins inhibit helicase loading outside G1 by phosphorylating key factors (e.g., Cdc6 degradation, Cdt1 inhibition).

   • This tight regulation ensures helicase loading is a once-per-cycle event.

Molecular Mechanism of Helicase Loading

• ORC binds to replication origins during G1, marking sites for helicase loading.

• Cdc6 and Cdt1 facilitate the recruitment and loading of the Mcm2-7 helicase complex around dsDNA.

• Mcm2-7 is loaded as an inactive double hexamer, preventing premature helicase activity.

• Upon entry into S phase, additional factors (e.g., Cdc45, GINS) activate the helicase to form the CMG complex, initiating DNA unwinding and replication fork formation.

Importance of Helicase Loading Timing

• Ensures genome stability by preventing multiple rounds of replication at the same origin.

• Coordinates DNA replication with cell cycle progression.

• Allows cells to respond to DNA damage or replication stress by controlling origin licensing.

Summary Table: Helicase Loading and Activation in Eukaryotic Cell Cycle

Conclusion

Helicase loading in eukaryotic DNA replication is a highly regulated event that occurs exclusively during the G1 phase of the cell cycle. This temporal restriction is crucial to ensure that each replication origin is licensed once per cycle, preventing re-replication and maintaining genomic integrity. The process involves the coordinated action of ORC, Cdc6, Cdt1, and the Mcm2-7 helicase complex, setting the stage for helicase activation and DNA synthesis during S phase.