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Updated: Sep 17, 2025

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis
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Pol θ-mediated end-joining uses microhomologies containing mismatches

Yuzhen Li1, Ngoc K Dang1, Wei He1

  • 1Department of Epigenetics and Molecular Carcinogenesis, MD Anderson Cancer Center, Houston, TX, USA.

Nature Communications
|July 2, 2025
PubMed

Insights

DNA polymerase theta (Pol θ) repairs DNA by using short microhomologies. This study reveals Pol θ prefers mismatched microhomologies, influencing cancer mutation analysis.

Area of Science:

  • Molecular Biology
  • Genetics
  • Cancer Research

Background:

  • DNA polymerase theta (Pol θ) is crucial for repairing DNA double-strand breaks.
  • Understanding Pol θ's mechanism is vital as some cancers depend on it for survival.
  • Microhomology selection by Pol θ influences genomic stability and mutation patterns.

Purpose of the Study:

  • To investigate the microhomology selection process used by purified human Pol θ during DNA end-joining.
  • To determine the factors influencing Pol θ's choice of microhomologies.
  • To refine the definition of microhomology in the context of Pol θ activity.

Main Methods:

  • Utilized purified human Pol θ for in vitro end-joining assays.
  • Employed DNA sequencing to analyze products generated from diverse oligonucleotide libraries.
  • Investigated the role of sequence features near the 3' end in microhomology selection.

Main Results:

  • Pol θ preferentially selects short, internal microhomologies within 15 nucleotides of the single-stranded DNA terminus.
  • Selected microhomologies are frequently interrupted by mismatches.
  • Base pairing within 6 nucleotides of the 3' end significantly impacts microhomology choice.
  • Bidirectional synthesis is not required for Pol θ-mediated end-joining initiation.

Conclusions:

  • Pol θ's preference for mismatched microhomologies necessitates a revised definition of microhomology.
  • Findings advance the understanding of DNA repair mechanisms and Pol θ's role in genomic integrity.
  • This research has implications for analyzing cancer genomes and developing targeted therapies.

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