Magnetic Tweezers and Fluorescence to study DNA: protein interactions

Resum

The last decade has witnessed howinstrumental development has pushed the limits of singlemolecule biophysics. An e ort has been made in combining force spectroscopy and uorescence microscopy giving rise to a range of sophisticated setups that permit manipulating biomolecules at the same time they can be visualized by tagging them with uorophores. In this thesis, we have combined Magnetic Tweezers and Total Internal Re ection Fluorescence (TIRF) microscopy. This combination resulted in a system that allows applying a controlled force over several DNA molecules while their interactions with uorescently labelled proteins are simultaneously visualized. This dissertation provides descriptions of both techniques, as well as details on the design, construction and characterization experiments of such a hybrid setup. Our apparatus has also incorporated multistream laminar ow technology for a precise fast exchange of reagents in the sample. The setup has been applied to the study of the binding and condensation mechanisms of the protein ParB from Bacillus subtilis. ParB is involved in the segregation apparatus of low copy number plasmids and it is a model for chromosome segregation in many bacteria. Although its role in DNA condensation by network formation mechanism has been previously described, its binding mechanism remained elusive. This thesis has contributed to understand its DNA binding as well as the role of the C-terminal domain (CTD) of the protein in DNA condensation inhibition. Our novel experimental approach evidenced the dynamic nature of ParB showing a fast exchange between DNA-bound ParB and protein in the surrounding media. Additionally, our study of the e ect of the CTD of ParB in binding and condensation allowed us to validate a model in which the CTD of ParB blocks ParB network formation by heterodimerization with the full-length protein, which remains bound to the DNA. A similar combined setup has been applied to the study of Cascade from Streptococcus thermophilus under the supervision of Prof. Dr. Ralf Seidel at Universität Leipzig, during a three-month predoctoral internship. The CRISPR-Cas systems are RNA-protein ribonucleoprotein complexes that constitute an adaptative immunity system which protects prokaryotes from foreign nucleic acids. The dynamics of the so-called R-loop formation has been studied at the single-molecule level, but the experimental approach lacked a characterization on protein binding or conformational changes prior to R-loop formation. In the present research work we aim to obtain correlated Magnetic Tweezers and uorescence data to provide insight in the binding mechanism of Cascade. Although preliminary, results presented here helped towards the obtention of correlated measurements, which demonstrated that R-loop formation takes place almost instantaneously after Cascade binding.