Structural basis for Polθ-helicase DNA binding and microhomology-mediated end-joining

Area of Science:

• Molecular Biology
• Genomics
• Structural Biology

Background:

• DNA double-strand breaks (DSBs) threaten genomic integrity and can lead to cancer.
• Homologous recombination (HR) and non-homologous end joining (NHEJ) are primary DSB repair pathways.
• DNA polymerase theta (Polθ) is essential for microhomology-mediated end joining (MMEJ) in HR-deficient cells, presenting a therapeutic target.

Purpose of the Study:

• To elucidate the molecular mechanisms of Polθ-mediated microhomology-mediated end joining (MMEJ).
• To provide structural insights into the Polθ helicase domain's function in DSB repair.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was used to determine structures of the Polθ helicase domain (Polθ-hel).
• Structures were obtained in complex with DNA substrates featuring 3'-single-stranded DNA (ssDNA) overhangs.

Main Results:

• Cryo-EM structures reveal sequential conformations of Polθ-hel during DNA binding, microhomology searching, and annealing.
• Stepwise conformational changes in Polθ-hel subdomains and its dimeric state are critical for aligning 3'-ssDNA overhangs.
• These structural dynamics facilitate microhomology search and annealing essential for MMEJ.

Conclusions:

• The study identifies key molecular switches within Polθ-hel that regulate the MMEJ process.
• These findings provide a structural basis for understanding Polθ-mediated DSB repair.
• The research lays the foundation for developing targeted therapies against Polθ-hel in cancer treatment.