Background
DNA polymerase θ (Pol θ) is a key enzyme in the alternative end-joining (alt-EJ) DNA repair pathway. In cancers with BRCA1/2 mutations (which disable homologous recombination repair), cells become dependent on Pol θ for survival — making it a promising synthetic lethality target.
Novobiocin, a known antibiotic, was discovered to inhibit Pol θ and show anti-cancer activity in BRCA-mutant tumors. However, the structural basis for this inhibition was unknown.
The Challenge
- Small protein-ligand complex: The helicase domain is ~50 kDa
- Ligand identification: Visualizing the small molecule binding site at cryo-EM resolution
- Mechanistic understanding: How does novobiocin block ATP hydrolysis?
Shuimu's Approach
- Optimized construct design: Truncated helicase domain for improved homogeneity
- Complex stabilization: Incubation with novobiocin and ATP analogs
- High-resolution data collection: 300kV Titan Krios with K3 detector
- Focused refinement: Mask-based local refinement to improve ligand density
- First structure of Pol θ helicase domain bound to novobiocin
- Revealed the ATP-competitive binding mode in the helicase active site
- Identified key hydrogen bonds and hydrophobic interactions
- Explained the selectivity of novobiocin for Pol θ over other helicases
Results
Impact
This structure enables rational design of more potent and selective Pol θ inhibitors as alternatives to PARP inhibitors for treating BRCA-mutant cancers. Several pharmaceutical companies are now developing Pol θ-targeting drugs based on structural insights from this work.
Publication
Cryo-EM structure of DNA polymerase θ helicase domain in complex with inhibitor novobiocin — bioRxiv, 2023. Read the paper →