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Unrepaired base excision repair intermediates in template DNA strands trigger replication fork collapse and PARP

Almudena Serrano-Benitez1,2, Sophie E Wells3, Lylah Drummond-Clarke3

  • 1Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK.

The EMBO Journal
|July 26, 2023
PubMed

Insights

DNA single-strand breaks (SSBs) cause chromosome breakage when present ahead of replication forks. This occurs in SSB repair-defective cells treated with 5-chloro-2'-deoxyuridine (CldU), suggesting clinical utility.

Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • DNA single-strand breaks (SSBs) are critical lesions that can lead to replication stress and genomic instability.
  • The precise mechanism by which SSBs induce chromosome breakage, particularly their location relative to replication forks, remains incompletely understood.

Purpose of the Study:

  • To investigate whether SSBs located behind or ahead of replication forks induce chromosome breakage.
  • To elucidate the role of uracil DNA glycosylase (UNG) and 5-chloro-2 -deoxyuridine (CldU) in SSB-induced DNA damage and cytotoxicity.

Main Methods:

  • Utilized SSB repair-defective human cells (PARP or XRCC1 deficient) with enhanced sensitivity to CldU.
  • Assessed chromosome breakage, sister chromatid exchange, and cytotoxicity following CldU treatment.
  • Investigated the role of uracil DNA glycosylase (UNG) activity during S phase.

Main Results:

  • CldU treatment induced chromosome breakage, sister chromatid exchange, and cytotoxicity in SSB repair-defective cells.
  • The observed DNA damage and cytotoxicity were dependent on S phase UNG activity.
  • CldU incorporation was cytotoxic only in the subsequent cell cycle when present in template DNA, specifically when UNG-induced SSBs occurred in template strands ahead of replication forks.
  • BRCA-defective cells exhibited hypersensitivity to CldU, alone or with PARP inhibitors.

Conclusions:

  • SSBs ahead of replication forks, rather than behind them, are the primary trigger for replication fork collapse and chromosome breakage.
  • CldU-induced DNA damage and cytotoxicity are mediated by UNG activity on template DNA strands.
  • The hypersensitivity of BRCA-defective cells to CldU suggests potential clinical applications for CldU, possibly in combination with PARP inhibitors, for cancer therapy.
Keywords:
DNA repairDNA replication stressDNA strand breaksPARP-1genome instability

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