Domain interactions affecting human DNA topoisomerase I catalysis and camptothecin sensitivity

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• DNA topoisomerase I (Top1p) is a crucial enzyme for DNA replication and transcription, targeted by the anticancer drug camptothecin (Cpt).
• Mutations conferring Cpt resistance can alter Top1p structure and function, providing insights into drug-enzyme interactions.
• Understanding these mutations is key to developing more effective Top1p-targeting therapies.

Purpose of the Study:

• To identify structural features of human Top1p critical for Cpt binding and cytotoxicity.
• To develop a novel yeast genetic screen for isolating catalytically active, Cpt-resistant Top1p mutants.
• To elucidate the relationship between Top1p mutations, Cpt resistance, and enzyme catalysis.

Main Methods:

• A novel yeast genetic screen was employed to isolate human top1 mutants exhibiting catalytic activity but resistance to Cpt.
• Specific Top1p mutations, including substitutions at Gly363, were introduced and analyzed.
• Biochemical assays and molecular modeling were used to investigate enzyme activity and structural interactions.

Main Results:

• The screen yielded Top1p mutants with substitutions at Gly363 (Ser or Val) that suppressed Cpt sensitivity.
• Different Gly363 substitutions exhibited varying effects on suppressing Cpt sensitivity and maintaining catalytic activity.
• Mutations at Gly363 and Tyr723 influence Cpt binding site formation and enzyme catalysis.

Conclusions:

• Interactions between conserved Top1p domains, specifically the "lip" region (Gly363) and active site tyrosine (Tyr723), are critical for Cpt binding and catalysis.
• Gly363 substitutions can modulate Cpt resistance and Top1p catalytic activity.
• These findings provide a structural basis for understanding Cpt resistance and inform the design of novel Top1p inhibitors.