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Updated: Jun 23, 2025

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis
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Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

Published on: June 19, 2018

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Dynamic stem-loop extension by Pol θ and templated insertion during DNA repair

Denisse Carvajal-Maldonado1, Yuzhen Li1, Mark Returan1

  • 1Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Center, Houston, Texas, USA.

The Journal of Biological Chemistry
|June 14, 2024
PubMed

Insights

DNA polymerase theta (Pol θ) repairs cancer cell DNA breaks via theta-mediated end joining (TMEJ). This study reveals Pol θ

Area of Science:

  • Molecular Biology
  • Genetics
  • Cancer Research

Background:

  • Theta-mediated end joining (TMEJ) is a DNA repair pathway crucial for cancer cell survival when other pathways are compromised.
  • Human DNA polymerase theta (Pol θ) plays a role in TMEJ, but its substrate preferences and repair mechanisms are not fully understood.

Purpose of the Study:

  • To investigate the substrate specificity and mechanism of human DNA polymerase theta (Pol θ) in DNA repair.
  • To elucidate the role of Pol θ in theta-mediated end joining (TMEJ) and its contribution to cancer cell survival.

Main Methods:

  • In vitro reconstitution of TMEJ using purified human Pol θ.
  • Analysis of Pol θ's ability to extend ssDNA and RNA substrates using various primer-template configurations.
  • Investigation of the impact of replication protein A (RPA) and Pol θ-specific residues on TMEJ activity.

Main Results:

  • Pol θ can extend both ssDNA and RNA substrates via unimolecular stem-loop synthesis, initiated by minimal base pairing.
  • Pol θ exhibits flexibility in primer-template interactions, leading to diverse sequence outcomes and low-fidelity repair under imbalanced nucleotide conditions.
  • Unimolecular stem-loop synthesis by Pol θ competes with bimolecular end joining and is partially inhibited by RPA.
  • Pol θ-specific residues are essential for rapid stem-loop synthesis, and related reactions can be catalyzed by Pol η and Pol λ.
  • Reconstituted TMEJ reactions incorporated insertions derived from stem-loop extensions, explaining inverted repeat sequences in repair events.

Conclusions:

  • Human Pol θ possesses a unique ability for stem-loop synthesis from minimally paired primers, contributing to TMEJ.
  • Pol θ's versatile repair mechanisms, including stem-loop synthesis, play a significant role in TMEJ and may contribute to genomic instability in cancer.
  • Understanding Pol θ's function in TMEJ provides insights into cancer cell survival mechanisms and potential therapeutic targets.
Keywords:
DNA polymeraseDNA repairDNA synthesisRNA synthesisprotein–DNA interaction

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