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

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.