A Practical Site-specific Method for the Detection of Bulky DNA Damages

Hiba Hassanain, Dana Tseitline, Tamar Hacohen, Adi Yifrach, Ayala Kirshenbaum, Bar Lavi, Avital Parnas, Sheera Adar*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Helix-distorting DNA damages block RNA and DNA polymerase, compromising cell function and fate. In human cells, these damages are removed primarily by nucleotide excision repair (NER). Here, we describe damage-sensing PCR (dsPCR), a PCR-based method for the detection of these DNA damages. Exposure to DNA damaging agents results in lower PCR signal in comparison to non-damaged DNA, and repair is measured as the restoration of PCR signal over time. We show that the method successfully detects damages induced by ultraviolet (UV) radiation, by the carcinogenic component of cigarette smoke benzo[a]pyrene diol epoxide (BPDE) and by the chemotherapeutic drug cisplatin. Damage removal measured by dsPCR in a heterochromatic region is less efficient than in a transcribed and accessible region. Furthermore, lower repair is measured in repair-deficient knock-out cells. This straight-forward method could be applied by non-DNA repair experts to study the involvement of their gene-of-interest in repair. Furthermore, this method is fully amenable for high-throughput screening of DNA repair activity.

Original languageAmerican English
Article number168450
JournalJournal of Molecular Biology
Volume436
Issue number6
Early online date20 Jan 2024
DOIs
StatePublished - 15 Mar 2024

Bibliographical note

Publisher Copyright:
© 2024 Elsevier Ltd

Keywords

  • DNA adducts
  • DNA damage
  • DNA repair
  • PCR
  • nucleotide excision repair

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