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

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.