su min (violet) hong

read_status: annotated
type: paper
title: "Replication Fork Activation Is Enabled by a Single-Stranded DNA Gate in CMG Helicase"
year: 2019
authors:
- "Wasserman, Michael R."
- "Schauer, Grant D."
- "O’Donnell, Michael E."
- "Liu, Shixin"
journal: "Cell"
doi: "10.1016/j.cell.2019.06.032"
url: "https://linkinghub.elsevier.com/retrieve/pii/S0092867419307354"
project:
  - IGP
system:
  - replication_fork
  - replisome
  - chromatin
  - single_stranded_dna
  - double_stranded_dna
disease:
  - replication_stress
  - dna_damage
genes:
  - mcm2
  - mcm3
  - mcm4
  - mcm5
  - mcm6
  - mcm7
  - cdc45
  - gins
  - mcm10
  - rpa
processes:
  - helicase_loading
  - dna_unwinding
  - replisome_assembly
  - fork_stalling
  - fork_restart
  - strand_passage
  - diffusion_on_dna
methods:
  - single_molecule_fluorescence
  - optical_tweezers
  - correlative_force_fluorescence_microscopy
  - bulk_helicase_assay
  - in_vitro_replisome_reconstitution
key_findings:
  - ss_to_ds_dna_transition
  - ds_to_ss_dna_transition
  - helicase_ring_gating
  - replisome_preservation
  - polymerase_coupling_dependence
limitations:
  - gate_structure_unresolved
  - in_vivo_validation_limited
  - chromatin_context_not_tested
relevance:
  - replication_restart_mechanism
  - conceptual_shift_in_helicase_dynamics
  - replisome_stress_response
concepts:
  - ssDNA_gating_in_ring_helicases
  - replisome_preservation
  - helicase_mode_switching
tags:
  - dna_replication
  - helicase
  - replication_stress
  - single_molecule

Abstract

The eukaryotic replicative helicase CMG is a closed ring around double-stranded (ds)DNA at origins yet must transition to single-stranded (ss)DNA for helicase action. CMG must also handle repair intermediates, such as reversed forks that lack ssDNA. Here, using correlative single-molecule fluorescence and force microscopy, we show that CMG harbors a ssDNA gate that enables transitions between ss and dsDNA. When coupled to DNA polymerase, CMG remains on ssDNA, but when uncoupled, CMG employs this gate to traverse forked junctions onto dsDNA. Surprisingly, CMG undergoes rapid diffusion on dsDNA and can transition back onto ssDNA to nucleate a functional replisome. The gate—distinct from that between Mcm2/5 used for origin loading—is intrinsic to CMG; however, Mcm10 promotes strand passage by enhancing the affinity of CMG to DNA. This gating process may explain the dsDNAto-ssDNA transition of CMG at origins and help preserve CMG on dsDNA during fork repair.

Key Findings

• CMG helicase can reversibly switch between ssDNA and dsDNA binding modes

• A transient ssDNA gate in CMG enables strand passage without disassembly

• When polymerase-coupled, CMG remains on ssDNA and supports processive replication

• Polymerase uncoupling triggers CMG transition to a diffusive dsDNA mode

• Diffusive CMG can re-enter a fork and re-initiate replisome assembly

• Mcm10 is required to retain CMG on DNA during gating and mode switching

• Mcm2/5 interface is not the ssDNA gate used during fork transitions

Methods

• Correlative single-molecule fluorescence + optical tweezers to track CMG on DNA

• Force-induced generation of ssDNA–dsDNA junctions mimicking replication forks

• Dual- and triple-color imaging of CMG, Mcm10, and RPA

• In vitro reconstituted replisome assays with labeled nucleotide incorporation

• Mean-square-displacement analysis to quantify helicase diffusion on dsDNA

Notes

• Introduces dynamic helicase behavior, not static fork association

• Reframes CMG as a reusable replisome scaffold rather than a one-use machine

• Provides a mechanistic explanation for fork restart without new origin firing

• Separates reversible mode switching from irreversible CMG unloading pathways

• Clarifies a long-standing question of how CMG transitions from dsDNA to ssDNA at origins

Figures / Data

-Figure 1: CMG undergoes ATP-dependent directional translocation on ssDNA

• Figure 2: Mcm10 is essential for efficient CMG ssDNA loading

• Figure 3: CMG preferentially loads at ss–ds DNA junctions

• Figure 4: Fork-loaded CMG nucleates active replisome assembly

• Figure 5: CMG switches to rapid diffusion on dsDNA when uncoupled

• Figure 6: Mcm10 is required for CMG mode switching and DNA retention

• Figure 7: Conceptual model of replisome preservation and restart

Limitations

• Physical location and structure of the ssDNA gate remain unknown

• Experiments performed on naked DNA, not nucleosome-dense chromatin

• Limited direct in vivo visualization of CMG diffusion during stress

• Checkpoint signaling effects on gating not mechanistically dissected