Periodic Reporting for period 4 - Mechan-of-Chromo (Unfolding the Mechanism of Chromosome Cohesion and Condensation using Single-Molecule Biophysical Approaches) Reporting period: 2020-12-01 to 2021-11-30 Summary of the context and overall objectives of the project Chromosome condensation, cohesion and segregation are fundamental biological processes whose failure leads to cell death, cancer and developmental defects. However, unravelling the mechanisms of DNA cohesion and condensation is a particularly challenging task because these processes are fundamentally mechanical in nature and thus particularly difficult to monitor using conventional biochemistry. Instead, single-molecule manipulation and imaging techniques are particularly well-suited for monitoring DNA cohesion and condensation, because they allow us to manipulate individual DNA molecules, measure forces, and add proteins and buffer solutions in a carefully controlled manner.At the core of these processes are the Structural Maintenance of Chromosome (SMC) proteins that mediate the global folding of the chromosome and stabilize the higher-order chromatin architecture by bringing distant DNA sequences together. Many disparate working models have attempted to explain how SMC complexes may tether (cohesion) and/or pack (condensation) DNA in a manner dependent on ATP binding/hydrolysis and higher-order interactions between individual SMC complexes. In this project, we aim to understand how SMC complexes contribute to chromosome organisation by recapitulating cohesion and condensation reactions in vitro one molecule at a time.The overall aims of this interdisciplinary project were two-fold:1) To develop beyond state-of-the-art instrumentation and novel biophysical assays to monitor DNA condensation and cohesion combining Magnetic Tweezers, fluorescence imaging and optical trapping.2) To assess the role of SMC proteins, SMC loaders, ATP binding/hydrolysis, and kleisin subunits in chromosome cohesion and condensation assays using these newly-devised biophysical approaches.We have successfully built a new instrument that include single-molecule manipulation based on magnetic tweezers, multichannel flow cells, lateral pulling of magnetic beads, and TIRF microscopy. We have also acquired for purpose of the project a dual optical tweezers setup combined with confocal microscopy. New methods for these setups were implemented for data acquisition and analysis with the aim to investigate the activity of SMC and SMC-related proteins involved in DNA processing and chromosome organisation. Eucaryotes contain three distinct SMC complexes known as cohesin, condensin, and Smc5/6. During the course of the project, the SMC field was revolutionised by the purification of active holocomplexes for cohesin and condensin and studied with biophysical techniques, which have resulted in a number of high impact publications in the last couple of years. We reported the first purification of the active Smc5/6 holocomplex, the third eukaryotic SMC complex, and showed for the first time that these complexes can entrap structure-specific DNA substrates and compact DNA in an ATP-dependent manner. Additionally, in an independent work, we showed at the single molecule level the specific binding of ParB to parS and the loading to non-parS DNA using parS as the entry gate to the DNA. The process was CTP-binding dependent but did not require the hydrolysis of CTP. Importantly, we discovered that this mechanism facilitates DNA condensation at nanomolar concentration of ParB, a process exclusively dependent on the presence of CTP and parS. Finally, we implemented a new methodology based on Molecular Dynamics to stretch nucleic acids and study the mechanical properties of DNA and RNA. We provided an explanation for the long-standing question of why a molecule of dsDNA overwinds when stretched in contrast to the opposite behaviour shown for dsRNA, and explained the three-fold softer stretching constant exhibited by dsRNA compared to dsDNA. This novel methodology contributed to advance the frontier of knowledge of the mechanics of nucleic acids (see list of publications). Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Condensation and cohesion of DNA molecules involve both inter- and intra-molecular interactions between segments of DNA. The study of these phenomena using single-molecule techniques requires the design of bespoke instrumentation, and methods for data acquisition and analysis.We have built a new instrument combining magnetic tweezers, multichannel flow cells, lateral pulling of magnetic beads, and TIRF microscopy. This instrumentation allows us to apply novel methods to study DNA-protein interactions and in particular the action of proteins involved in condensation and/or cohesion of DNA which link distant segments of DNA in cis or in trans. We have implemented novel methods to study DNA condensation by chromosome related proteins. These methodologies have allowed us to underpin the mechanism of DNA condensation by ParB proteins and to unravel the kinetics of its interaction with DNA. Moreover, using these instruments we reported for the first time condensation activity of the Smc5/6 holocomplex, the third SMC complex studied with biophysical techniques. We have also developed novel methodologies employing Molecular Dynamics (MD) simulations to understand the behaviour of nucleic acids under tension and torsion. These novel methods allowed us to stretch nucleic acids and in-silico study the mechanical properties of DNA and RNA. We explained why a molecule of dsDNA overwinds when stretched in contrast to the opposite behaviour shown for dsRNA, and exhibits a stretching constant three-fold softer than dsRNA. We also introduced the concept of crookedness in nucleic acids, which is relevant for local bending of DNA and RNA; and described the presence of AU tracts in dsRNA and their role in RNA-protein interactions. By the end of the project, our research has led to over 20 publications in highly-recognized journals. The team of the project has contributed around 80-100 dissemination activities and 10 awards and/or institutional recognitions are reported. Importanlty, this grant allowed the completion of 4 PhD projects and opportunities to about 15 students through short research expeditions or Master projects. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The instruments we developed are state-of-art technology suitable to investigate DNA-protein interactions at the single-molecule level. These required the development of novel methodologies for data acquisition and analysis.