Duplex DNA and BLM regulate gate opening by the human TopoIIIα-RMI1-RMI2 complex

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Topoisomerase IIIα, complexed with RMI1 and RMI2 as the TRR complex, is crucial for resolving DNA replication and recombination intermediates.
• The TRR complex functions alongside the BLM helicase in these processes.
• The precise mechanism of the TRR catalytic cycle, particularly its protein-mediated single-stranded (ss)DNA gate, remains incompletely understood.

Purpose of the Study:

• To investigate the detailed catalytic mechanism of the TRR complex.
• To elucidate the function and dynamics of the TRR-ssDNA gate.
• To explore the influence of DNA binding and the BLM helicase on TRR activity.

Main Methods:

• Utilized optical tweezers and fluorescence microscopy to probe TRR's catalytic steps.
• Measured the size of the TRR-ssDNA gate.
• Visualized the binding of ssDNA and double-stranded (ds)DNA to the TRR-ssDNA gate and subsequent catenation.

Main Results:

• Demonstrated that TRR forms an open ssDNA gate of 8.5 ± 3.8 nm.
• Directly visualized the binding of a second ssDNA or dsDNA molecule to the open gate, leading to catenation.
• Observed that dsDNA binding expands the gate size by approximately 16%, and BLM alters gate mechanical flexibility.

Conclusions:

• Revealed significant plasticity in the TRR-ssDNA gate size.
• Suggests that TRR-mediated transfer of dsDNA is a more relevant in vivo mechanism than previously thought.
• Provides new insights into the mechanistic details of DNA topological management by the TRR complex.