The sequence-dependence of biomolecular interactions involving nucleic acids and proteins is essential for numerous processes inside the cell. Insights into the underlying molecular mechanisms have... Show moreThe sequence-dependence of biomolecular interactions involving nucleic acids and proteins is essential for numerous processes inside the cell. Insights into the underlying molecular mechanisms have been obtained using various biochemical and biophysical methods on two different levels — bulk and single-molecule. These have complemented each other as single-molecule studies excel in observing multi-state dynamic interactions, but perform only at low throughput; while bulk studies can probe many different sequences simultaneously, but providing limited kinetic information. To unite the strengths of both levels, we developed high-throughput Single-molecule Parallel Analysis for Rapid eXploration of Sequence space (SPARXS), that allows the study of molecular structure, kinetics and interactions for thousands of different sequences simultaneously at the single-molecule level. We, for the first time, combine single-molecule fluorescence with next-generation Illumina sequencing. As a proof of principle we apply SPARXS to study the sequence-dependent kinetics of the four-way DNA Holliday junction, occurring among others during homologous recombination. Using SPARXS we observe the dynamic behavior of 120,000 Holliday junction molecules covering 3750 distinct core sequences, a result unattainable with previous techniques. Overall, the mechanistic insights obtained using SPARXS will give an entirely new view on the relationship between sequence, structure and function. Show less
This thesis investigates the characteristics and the mechanisms of DNA replicative proteins through the use of biochemistry, single molecule light mycroscopy and Cryo-EM.
Ewing sarcoma (ES) is a special type of bone cancer, first described by Dr. James Ewing in his paper __Diffusive endothelioma of bone__. Today Ewing sarcoma represents the second most common bone... Show moreEwing sarcoma (ES) is a special type of bone cancer, first described by Dr. James Ewing in his paper __Diffusive endothelioma of bone__. Today Ewing sarcoma represents the second most common bone cancer among adolescents and young adults. Contrary to the positive achievement in treatment of localized tumors, the long-term (5-years) survival for Ewing sarcoma patients with metastasis, however, remain below the 30% mark. In this thesis a report on experimental work aiming for a better understanding of the mechanisms underlying Ewing sarcoma metastasis is presented. Two distinct mechanisms are investigated: (1) a biochemical approach in which the initial steps in the CXCR4 signaling cascade are followed, and (2) a biophysical approach in which the guidance of Ewing sarcoma metastasis by the stiffness of their microenvironment is demonstrated. The results presented in this thesis provide deeper insights into the mechanisms controlling signaling of the chemokine receptor CXCR4 and into the role of the micro-environment in Ewing sarcoma cells behavior.Through various experimental approaches it was shown that both biochemical and biophysical guidance control how Ewing sarcoma develops into its distinct metastatic phenotype. Show less
Since the discovery of the right-handed helical structure of DNA, 61 years have passed. The DNA molecule, which encodes genetic information, is also found twisted into coils. This extra twist of... Show moreSince the discovery of the right-handed helical structure of DNA, 61 years have passed. The DNA molecule, which encodes genetic information, is also found twisted into coils. This extra twist of the helical structure, called supercoiling, plays important roles in both DNA compaction and gene regulation. The DNA in eukaryotic cells is packaged into chromatin. Using single-molecule force spectroscopy, I resolved force/torque induced structural changes of DNA and chromatin fibers. I showed that the structural changes of chromatin fibers can be described by four conformations. I showed for the first time the folding and unfolding of a chromatin fiber under torsion. Th e anisotropic response of chromatin fibers to supercoiling reflects its leftŸ-handed chirality. These findings give a detailed structural insight of a supercoiled chromatin fiber, yielding a better understanding of the response of chromatin during transcription Show less
Understanding of chromatin organization and compaction in Archaea is currently limited. The genome of several megabasepairs long is folded by a set of small chromatin proteins to fit into the... Show moreUnderstanding of chromatin organization and compaction in Archaea is currently limited. The genome of several megabasepairs long is folded by a set of small chromatin proteins to fit into the micron-sized cell. A first step in understanding archaeal chromatin organization is to study the action of individual chromatin proteins on DNA. Characterization of the architectural properties of these proteins is essential to understand how they shape and modulate the archaeal genome. This thesis describes the biophysical characterization of several chromatin proteins from the crenarchaeal model organism Sulfolobus solfataricus: Cren7, Sul7, Alba and Sso10a. The architectural properties of these proteins resemble those of their bacterial counterparts, suggesting that they could play a similar role in chromatin organization and global gene regulation. Show less
This thesis discusses the purification and activities of the SARS-coronavirus (SARS-CoV) RNA-dependent RNA polymerases (RdRps) nsp12 and nsp(7+8). The first is a large monomeric RdRp, whose... Show moreThis thesis discusses the purification and activities of the SARS-coronavirus (SARS-CoV) RNA-dependent RNA polymerases (RdRps) nsp12 and nsp(7+8). The first is a large monomeric RdRp, whose stability is greatly influenced by N-terminal additions. In contrast, the latter RdRp is a remarkable hexadecameric complex that is capable of both de novo initiation and primer-extension. In addition these RNA polymerase activities, the thesis also expatiates upon the calibration of magnetic tweezers for force measurements and the dynamics of the equine arteritis virus (EAV) RNA helicase as function of NTP hydrolysis. Show less
