Faithful chromosome segregation requires that the sister chromatids be disjoined completely. Defective disjunction can lead to the persistence of histone-free threads of DNA known as ultra-fine bridges (UFBs) that connect the separating sister DNA molecules during anaphase. UFBs arise at specific genomic loci and can only be visualized by detection of associated proteins such as PICH, BLM, topoisomerase IIIα, and RPA. However, it remains unknown how these proteins work together to promote UFB processing. We used a combination of ensemble biochemistry and new single-molecule assays to reconstitute key steps of UFB recognition and processing by these human proteins in vitro. We discovered characteristic patterns of hierarchical recruitment and coordinated biochemical activities that were specific for DNA structures modeling UFBs arising at either centromeres or common fragile sites. Our results describe a mechanistic model for how unresolved DNA replication structures are processed by DNA-structure-specific binding factors in mitosis to prevent pathological chromosome nondisjunction.

High-speed hydrodynamic trapping enables combined surface-free force spectroscopy and fluorescence imaging of single DNA molecules at extreme forces.

Topoisomerases are essential enzymes that regulate DNA topology. Type 1A family topoisomerases are found in nearly all living organisms and are unique in that they require single-stranded (ss)DNA for activity. These enzymes are vital for maintaining supercoiling homeostasis and resolving DNA entanglements generated during DNA replication and repair. While the catalytic cycle of Type 1A topoisomerases has been long-known to involve an enzyme-bridged ssDNA gate that allows strand passage, a deeper mechanistic understanding of these enzymes has only recently begun to emerge. This knowledge has been greatly enhanced through the combination of biochemical studies and increasingly sophisticated single-molecule assays based on magnetic tweezers, optical tweezers, atomic force microscopy and Förster resonance energy transfer. In this review, we discuss how single-molecule assays have advanced our understanding of the gate opening dynamics and strand-passage mechanisms of Type 1A topoisomerases, as well as the interplay of Type 1A topoisomerases with partner proteins, such as RecQ-family helicases. We also highlight how these assays have shed new light on the likely functional roles of Type 1A topoisomerases in vivo and discuss recent developments in single-molecule technologies that could be applied to further enhance our understanding of these essential enzymes.

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.

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