Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase θ

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Microhomology-mediated end-joining (MMEJ) is an alternative DNA double-strand break repair pathway.
• The precise mechanism of MMEJ remains largely unknown, despite its implication in cancer development.

Purpose of the Study:

• To elucidate the molecular mechanism of microhomology-mediated end-joining (MMEJ).
• To investigate the role of human DNA polymerase theta (Polθ) in the MMEJ pathway.

Main Methods:

• Purification and biochemical assays of human DNA polymerase theta (Polθ).
• In vitro studies using DNA substrates with 3' single-strand overhangs and microhomology.
• Functional assays in human cells to assess MMEJ dependence on Polθ.

Main Results:

• Purified human Polθ directly performs MMEJ on DNA substrates with microhomology.
• MMEJ is dependent on Polθ in human cells.
• Polθ facilitates end-joining, microhomology annealing, and stabilization of the DNA synapse using opposing overhangs.
• Polθ shows a preference for 5'-terminal phosphorylated DNA, similar to nonhomologous end-joining polymerases.
• A conserved loop domain in Polθ is critical for MMEJ and higher-order structure formation.

Conclusions:

• Human DNA polymerase theta (Polθ) is a key enzyme in the MMEJ pathway.
• Polθ's mechanism involves facilitating DNA end-joining and annealing via microhomology.
• Polθ's unique structural features are essential for its role in MMEJ and DNA repair.