#	Pagina
attuale pagina	/open-h2020/projects/202576/results.html

Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - Mechan-of-Chromo (Unfolding the Mechanism of Chromosome Cohesion and Condensation using Single-Molecule Biophysical Approaches)

Teaser

Summary

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 are 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.

Work performed

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 (Madariaga-Marcos et al, eLife 2019; Fisher et al. eLife 2017; Madariaga-Marcos et al. Nanoscale 2018). We are now applying our new methods to study CcParB and other DNA condensation proteins. We expect these results to be published in 2020.

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 describe a novel DNA feature, named crookedness, which is relevant for local bending and for the type of DNA-protein interactions studied in the Mechan-of-Chromo project (MarÃn GonzÃ¡lez et al, PNAS 2017; MarÃn-Gonzalez et al PRL 2019). We are currently using our new methods based on MD simulations to study the mechanics of double stranded RNA and we expect these results to be published in 2019-2020.

By the end of the first 36 month period of the project, our research has led to 9 publications in highly-recognized journals. The team of the project has contributed in more than 65 dissemination activities and 7 awards and/or institutional recognitions are reported. Moreover, we have currently several papers under revision and are finalising the writing of other manuscripts. Progress beyond the state of the art and expected results until the end of the project
The instruments we are developing are state-of-art technology suitable to investigate DNA-protein interactions at the single-molecule level. Using these instruments, we have provided for the first time the basis of functioning of BsSpo0J, a key protein for the condensation of the bacterial chromosome and loading of the SMC complex. We will continue developing our instrumentation and new methodologies with the aim to exploit all the new features related with their fluorescent capabilities, as foreseen in the proposal. We expect that new experiments using fluorescently labeled SMC and ParB will help us to understand the mechanism of chromosome organisation in bacteria.

The instruments we are developing are state-of-art technology suitable to investigate DNA-protein interactions at the single-molecule level. Using these instruments, we have provided for the first time the basis of functioning of BsSpo0J, a key protein for the condensation of the bacterial chromosome and loading of the SMC complex. We will continue developing our instrumentation and new methodologies with the aim to exploit all the new features related with their fluorescent capabilities, as foreseen in the proposal. We expect that new experiments using fluorescently labeled SMC and ParB will help us to understand the mechanism of chromosome organisation in bacteria.

Final results

The instruments we are developing are state-of-art technology suitable to investigate DNA-protein interactions at the single-molecule level. Using these instruments, we have provided for the first time the basis of functioning of BsSpo0J, a key protein for the condensation of the bacterial chromosome and loading of the SMC complex. We will continue developing our instrumentation and new methodologies with the aim to exploit all the new features related with their fluorescent capabilities, as foreseen in the proposal. We expect that new experiments using fluorescently labeled SMC and ParB will help us to understand the mechanism of chromosome organisation in bacteria.