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CRISPR knockout genome-wide screens identify the HELQ-RAD52 axis in regulating the repair of cisplatin-induced

Lindsey M Pale1, Jude B Khatib1, Alexandra Nusawardhana1

  • 1Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA.

Nucleic Acids Research
|November 12, 2024
PubMed

Insights

Cisplatin resistance in cancer may involve single-stranded DNA (ssDNA) gaps. The HELQ-RAD52-BRCA pathway regulates these gaps, offering new targets for overcoming cisplatin resistance in BRCA-proficient cells.

Area of Science:

  • Molecular Biology
  • Cancer Research
  • DNA Repair

Background:

  • Platinum compounds like cisplatin are crucial cancer treatments, but their precise mechanisms and resistance pathways are not fully understood.
  • Accumulation of single-stranded DNA (ssDNA) gaps has emerged as a potential factor in cisplatin chemosensitivity, particularly in BRCA-mutated cancers.
  • The enzyme PRIMPOL catalyzes repriming, leading to ssDNA gap formation downstream of cisplatin lesions during DNA synthesis restart.

Purpose of the Study:

  • To investigate whether ssDNA gap accumulation directly confers cisplatin sensitivity in BRCA-proficient cells.
  • To identify genetic factors that mediate the cytotoxicity of cisplatin-induced ssDNA gaps using genome-wide screening.
  • To elucidate the regulatory axis controlling ssDNA gap processing and its impact on cisplatin sensitization.

Main Methods:

  • CRISPR-mediated genome-wide genetic screening was employed to identify genes modulating cisplatin sensitivity.
  • Overexpression of PRIMPOL in wild-type cells was used to study ssDNA gap accumulation.
  • Western blotting and DNA combing techniques were utilized to assess ssDNA gap formation and processing.

Main Results:

  • PRIMPOL overexpression in wild-type cells led to ssDNA gap accumulation but did not sensitize cells to cisplatin, indicating gap accumulation alone is insufficient for sensitivity in BRCA-proficient cells.
  • The helicase HELQ was identified as a suppressor of cisplatin sensitivity in PRIMPOL-overexpressing cells, correlating with reduced ssDNA accumulation.
  • RAD52 was identified as a mediator, promoting ssDNA gap accumulation via a BRCA-dependent mechanism.

Conclusions:

  • The HELQ-RAD52-BRCA axis plays a critical role in regulating ssDNA gap processing and influencing cisplatin sensitization.
  • Understanding this axis provides insights into mechanisms of cisplatin resistance and potential therapeutic strategies for BRCA-proficient cancers.
  • Targeting the HELQ-RAD52-BRCA pathway could offer novel approaches to enhance the efficacy of platinum-based chemotherapy.

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