Deciphering the human TopIIIα activity modulated by Rmi1 using magnetic tweezers

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Topoisomerases IA (TopoIAs) are essential enzymes found across all domains of life.
• Metazoa typically possess two TopoIAs: TopIIIα and TopIIIβ, which perform distinct cellular functions.
• TopIIIα plays a critical role in maintaining genome stability by resolving DNA topological issues during replication and recombination.

Purpose of the Study:

• To elucidate the detailed enzymatic cycle of Topoisomerase IIIα (TopoIIIα).
• To investigate the precise role of the partner protein Rmi1 in TopoIIIα function.
• To differentiate between intrinsic TopoIIIα catalytic properties and those modulated by Rmi1 using single-molecule approaches.

Main Methods:

• Utilized a single-molecule approach to analyze the Topoisomerase IIIα (TopoIIIα) enzymatic cycle.
• Investigated the interaction between TopoIIIα and its partner protein Rmi1.
• Characterized DNA binding kinetics and complex stabilization.

Main Results:

• The rate-limiting step for Topoisomerase IIIα (TopoIIIα) is DNA binding, which necessitates a single-stranded DNA region.
• TopoIIIα exhibits extended pauses during its catalytic cycle to stabilize the open cleaved complex.
• Rmi1 enhances TopoIIIα's efficiency by facilitating single-stranded DNA trapping and stabilizing the cleaved complex, promoting substrate discrimination.

Conclusions:

• Rmi1 is a critical partner protein that significantly boosts Topoisomerase IIIα (TopoIIIα) efficiency and substrate specificity.
• Rmi1's modulation of DNA binding and complex stabilization is essential for smooth in vivo DNA transaction processes.
• Understanding the TopoIIIα-Rmi1 interaction provides insights into genome stability mechanisms.