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Replication Fork Activation Is Enabled by a Single-Stranded DNA Gate in CMG Helicase.

Michael R Wasserman1, Grant D Schauer2, Michael E O'Donnell3

  • 1Laboratory of Nanoscale Biophysics and Biochemistry, The Rockefeller University, New York, NY 10065, USA.

Cell
|July 27, 2019
PubMed
Summary

The CMG helicase uses a novel DNA gate to transition between double-stranded and single-stranded DNA. This mechanism allows CMG to move onto double-stranded DNA during replication and repair, then return to single-stranded DNA to restart replication.

Keywords:
CMGDNA repairDNA replicationMcm10fork restartoptical tweezersorigin initiationreplisomesingle-molecule fluorescence

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Area of Science:

  • Molecular Biology
  • DNA Replication
  • Biophysics

Background:

  • The CMG helicase is essential for eukaryotic DNA replication, forming a ring around dsDNA at origins.
  • CMG must transition to ssDNA for helicase activity and handle repair intermediates like reversed forks.
  • Understanding CMG's DNA binding and translocation is crucial for comprehending replication and repair fidelity.

Purpose of the Study:

  • To investigate the mechanism by which CMG transitions between dsDNA and ssDNA.
  • To identify the structural components and regulatory factors involved in CMG's DNA substrate switching.
  • To elucidate how CMG handles complex DNA structures during replication and repair.

Main Methods:

  • Correlative single-molecule fluorescence and force microscopy.
  • In vitro assays to observe CMG-DNA interactions and translocation.
  • Biochemical analysis of CMG-DNA binding and strand passage.

Main Results:

  • CMG possesses an intrinsic ssDNA gate facilitating transitions between ssDNA and dsDNA.
  • Uncoupled CMG utilizes this gate to move onto dsDNA at forked junctions.
  • CMG exhibits rapid diffusion on dsDNA and can re-engage ssDNA to initiate replisome assembly.
  • Mcm10 enhances CMG's DNA binding affinity, promoting strand passage.

Conclusions:

  • The ssDNA gate is a key feature enabling CMG's dynamic DNA substrate engagement.
  • This gating mechanism explains CMG's dsDNA-to-ssDNA transition at origins and its role in fork repair.
  • CMG's ability to navigate and bind dsDNA is critical for maintaining replication fork stability and restart.