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Asymmetrical recognition and processing of double-strand breaks formed during DNA replication.

Matthew J Johnson1,2, Michael T Kimble1,2, Seoyeon Jeong1,2

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|December 16, 2025
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Summary

DNA end resection differs at replication-dependent double-strand breaks (DSBs). Mre11 protein preferentially binds blunt ends, promoting resection, while Ku protein avoids overhangs, allowing Mre11-independent repair pathways.

Keywords:
DNA double-strand breakDNA replicationKuMre11end resection

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Area of Science:

  • Molecular Biology
  • DNA Repair Mechanisms
  • Cellular Biology

Background:

  • DNA end resection is crucial for homology-directed repair of double-strand breaks (DSBs).
  • Resection at endonuclease-induced DSBs is well-studied, but less is known about DSBs arising during DNA replication.
  • Previous work established a system for generating replication-dependent DSBs in budding yeast using Cas9-D10A nickase.

Purpose of the Study:

  • To investigate the distinct mechanisms of DNA end resection at replication-dependent DSBs.
  • To elucidate the roles of key proteins like Mre11 and Ku in processing these specific types of DSBs.
  • To understand why replication-dependent DSBs are inefficiently repaired by nonhomologous end joining.

Main Methods:

  • Utilized a Cas9-D10A nickase system to create replication-dependent DSBs in budding yeast.
  • Analyzed the asymmetric nature of DSB ends, identifying blunt ends and 3' single-stranded DNA (ssDNA) overhangs.
  • Investigated the binding preferences of Mre11 and Ku to different DSB end types.
  • Assessed the requirement of Mre11 and resection pathways (Exo1, Dna2-Sgs1) for break repair.

Main Results:

  • Replication-dependent DSBs exhibit asymmetric ends: one blunt/near-blunt and one with a 3' ssDNA overhang.
  • Mre11 preferentially binds to blunt ends, displacing Ku and initiating resection.
  • DSB ends with 3' overhangs show minimal Ku binding and undergo Mre11-independent resection via Exo1 or Dna2-Sgs1.
  • Ku selectively binds blunt ends, potentially hindering nonhomologous end joining (NHEJ) at these breaks.

Conclusions:

  • DNA end resection pathways differ significantly between replication-dependent DSBs and canonical DSBs.
  • Ku protein's preferential binding to blunt ends may explain the poor repair of replication-dependent DSBs by NHEJ.
  • Findings reveal novel insights into DSB processing during DNA replication and its implications for genome stability.