Abstract
Topoisomerase IIIα is a type 1A topoisomerase that forms a complex with RMI1 and RMI2 called TRR in human cells. TRR plays an essential role in resolving DNA replication and recombination intermediates, often alongside the helicase BLM. While the TRR catalytic cycle is known to involve a protein-mediated single-stranded (ss)DNA gate, the detailed mechanism is not fully understood. Here, we probe the catalytic steps of TRR using optical tweezers and fluorescence microscopy. We demonstrate that TRR forms an open gate in ssDNA of 8.5 ± 3.8 nm, and directly visualize binding of a second ssDNA or double-stranded (ds)DNA molecule to the open TRR-ssDNA gate, followed by catenation in each case. Strikingly, dsDNA binding increases the gate size (by ~16%), while BLM alters the mechanical flexibility of the gate. These findings reveal an unexpected plasticity of the TRR-ssDNA gate size and suggest that TRR-mediated transfer of dsDNA may be more relevant in vivo than previously believed.
Highlights
Topoisomerase IIIα is a type 1A topoisomerase that forms a complex with RMI1 and RMI2 called TRR in human cells
For example, ScTopoIII forms a complex with Rmi[1] (RecQ-mediated genome instability protein 1)[20,21,22], while the human homologue, HsTopoIIIα forms a stable complex with co-factors RMI1 and RMI2
This is powerful because it provides a sensitive means to measure subtle conformational changes in protein structure that would be difficult to achieve based on single gate opening events
Summary
Topoisomerase IIIα is a type 1A topoisomerase that forms a complex with RMI1 and RMI2 called TRR in human cells. A conformational rearrangement occurs that results in the opening of the protein-ssDNA gate This allows a second DNA segment, generally referred to as the transported (T)-DNA, to enter into the central cavity of the enzyme. For example, ScTopoIII forms a complex with Rmi[1] (RecQ-mediated genome instability protein 1)[20,21,22], while the human homologue, HsTopoIIIα forms a stable complex with co-factors RMI1 and RMI2 (referred to as TRR) In both complexes, the presence of Rmi1/RMI1 has been shown to greatly enhance the (de)catenation activity of the core topoisomerase[20,21,22,23,24]. This has been attributed to the fact that the Rmi1/RMI1 co-factor introduces a looped structure into the central cavity of ScTopoIII/HsTopIIIα, respectively, that resembles the decatenation loop in EcTopoIII25
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