Solving knots with a twist: An optical tweezers approach to study proteins involved in ultrafine anaphase bridge dissolution

Due to the highly complex and dynamic nature of cell division, the occurrence of obstacles during this process is inevitable. One frequently occurring obstacle is the formation of ultrafine anaphase bridges (UFBs). UFBs are entangled DNA threads that link sister chromatids and become stretched when these sister chromatids are pulled apart during cell division. Eukaryotic organisms have evolved an intricate protein machinery to resolve UFBs. While the key proteins involved are known, many questions about the molecular mechanisms of UFB dissolution remain unanswered. This thesis addresses important questions regarding UFB dissolution mechanisms, focusing on two key UFB-associated proteins: the SNF2-like protein PICH and the Type 1A topoisomerase TopoIIIα, which exists in complex with RMI1 and RMI2, known as TRR. The foundation of this thesis is a combination of biochemical methods, the technique ‘dual-trap optical tweezers,’ and fluorescence microscopy. Dual-trap optical tweezers, a single-molecule technique, allows direct observation of individual molecules. For example, by monitoring single proteins interacting with single DNA substrates over time, dynamic and kinetic information can be directly obtained, which is difficult, if not impossible, to achieve using traditional ensemble assays. Moreover, dual-trap optical tweezers can apply tension to the DNA, simulating the stretching of UFBs during cell division, making it ideally suited for studying UFB processing in a highly detailed manner. One of the key steps in the application of single-molecule assays to study DNA-protein interactions is to biochemically construct tailor-made DNA substrates. This is demonstrated in Chapter 2, which exploits Gibson Assembly cloning to prepare DNA molecules containing nucleosome positioning sequences, in a highly controlled manner. Combined with fluorescence microscopy, this enables monitoring nucleosome dynamics while at the same time visualizing fluorescently-labeled proteins bound to the nucleosome array. Experiments revealed that although nucleosomes unwrap at tensions around 20 pN, histones, particularly histone H3, remain bound to the DNA even at tensions beyond 60 pN, suggesting that nucleosome unwrapping does not correlate with complete histone dissociation. Chapter 3 shows that by carefully designing the study strategy, long-standing questions can be answered. By using the method presented in Chapter 2, we studied the nucleosome remodeling properties of PICH. This revealed that PICH can invade nucleosome arrays at tensions of 3 pN and higher, and catalyzes nucleosome unwrapping most efficiently at tensions between approximately 5 and 10 pN. This catalytic activity requires ATP hydrolysis and is likely associated with DNA loop-extrusion by PICH oligomers. Using dual-color confocal fluorescence microscopy, we observed that , after nucleosome unwrapping, PICH can slide histones along the DNA. Together, these findings demonstrate that PICH is a tension-dependent nucleosome remodeler. Chapter 4 highlights the contribution of single-molecule assays into the understanding of the gate-opening dynamics and strand-passage mechanisms of Type 1A topoisomerases, as well as the mechanistic interactions of these enzymes with partner proteins, such as PICH. Chapter 4 furthermore discusses recent developments in single-molecule technologies that could be applied to further enhance our understanding of Type 1A topoisomerases. One application that could prove highly informative is the method called Optical DNA Supercoiling (ODS). This method allows the generation of negatively supercoiled DNA using dual-trap optical tweezers, as detailed in Chapter 5. Chapter 6 presents how ODS can uniquely monitor the supercoil relaxation activity of the TRR complex in real time. The results show that TRR exhibits bursts of activity, separated by pauses, and demonstrate that the burst size and pause duration are both tension-dependent. Strikingly, we observe that a single TRR complex can perform thousands of catalytic cycles without unbinding and that TRR remains bound to the DNA long after supercoil relaxation has completed.