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) are dangerous DNA lesions that can lead to genomic instability and cancer.
• Homologous recombination (HR) and non-homologous end joining (NHEJ) are primary DSB repair pathways.
• In HR-deficient cells, DNA polymerase theta (Polθ) is essential for DSB repair via microhomology-mediated end joining (MMEJ).

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 (Polθ-hel) during DNA repair.
• To identify potential therapeutic targets for Polθ in HR-deficient cancers.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was used to determine structures of the Polθ helicase domain.
• Structures were obtained for Polθ-hel in complex with DNA containing 3'-overhangs.
• Analysis of sequential conformations during DNA binding, microhomology searching, and annealing.

Main Results:

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

Conclusions:

• Polθ-hel contains essential molecular switches that orchestrate the MMEJ process for DSB repair.
• The structural insights provide a foundation for developing targeted therapies against Polθ-hel.
• Polθ is a promising synthetic lethal target in BRCA1/2-deficient cancers.