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Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay
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Translesion-synthesis-mediated bypass of DNA lesions occurs predominantly behind replication forks restarted by

Ashna Dhoonmoon1, Julia R Ambrose1, Sonal Garg1

  • 1Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.

Cell Reports
|February 27, 2025
PubMed

Insights

Translesion synthesis (TLS) polymerases like Polκ and Polη fill gaps after DNA replication forks restart. This process, dependent on PrimPol, prevents DNA damage from forming double-strand breaks.

Area of Science:

  • Molecular Biology
  • Genetics
  • DNA Repair

Background:

  • Translesion synthesis (TLS) bypasses DNA lesions, enabling DNA replication completion.
  • The timing of TLS relative to replication fork restart has been debated.

Purpose of the Study:

  • To investigate the recruitment of TLS polymerases Polκ and Polη to DNA adducts.
  • To determine the role of PrimPol in TLS and replication restart.
  • To elucidate the mechanism by which TLS polymerases prevent DNA double-strand breaks.

Main Methods:

  • Development of cell imaging techniques using proximity ligation.
  • Monitoring recruitment of Polκ and Polη to specific DNA adducts (BPDE, cisplatin).
  • Analysis of DNA damage accumulation in TLS polymerase-deficient cells.

Main Results:

  • TLS polymerase recruitment is adduct-specific (Polκ for BPDE, Polη for cisplatin).
  • PrimPol is essential for repriming and restarting stalled replication forks.
  • TLS deficiency leads to adduct-specific single-stranded DNA (ssDNA) gaps and double-strand breaks (DSBs).

Conclusions:

  • TLS primarily occurs behind the restarted replication fork to fill PrimPol-generated gaps.
  • TLS is crucial for preventing the conversion of ssDNA gaps into cytotoxic DSBs in a lesion-specific manner.
Keywords:
BPDECP: Molecular biologyDNA adductsPCNAPrimPolcisplatindouble-strand breakspolymerase etapolymerase kappareplication fork stabilityreplication stressssDNA gapstranslation synthesis

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