Connecting the dots: key insights on ParB for chromosome segregation from single-molecule studies

Bacterial cells require DNA segregation machinery to properly distribute a genome to both daughter cells upon division. The most common system involved in chromosome and plasmid segregation in bacteria is the ParABS system. A core protein of this system - partition protein B (ParB) - regulates chromosome organization and chromosome...

What is all this fuss about Tus?: Comparison of recent findings from biophysical and biochemical experiments

Synchronizing the convergence of the two-oppositely moving DNA replication machineries at specific termination sites is a tightly coordinated process in bacteria. In Escherichia coli, a “replication fork trap” – found within a chromosomal region where forks are allowed to enter but not leave – is set by the protein–DNA roadblock Tus–Ter. The...

Single-molecule approaches to unravel the mechanism of SMC proteins

Every cell deals with the challenge of organising its DNA. First, the DNA needs to be compacted in size by several orders of magnitude. For example, in each human cell, 2 meters of DNA need to fit inside a micron-sized cell nucleus. Second, the DNA needs to stay accessible for cellular processes such as transcription and replication. To achieve...

Characterization of torque-spectroscopy techniques for probing rotary nanomotors

This thesis describes developments in the characterization of torque-spectroscopy techniques, in particular magnetic and optical tweezers, with the goal of employing these techniques in studies on the bacterial flagellar motor of Escherichia coli.

Assembling a single-molecule view on nucleosome dynamics

The main focus of this thesis is a better understanding of the basic compaction mechanism of our DNA using multiple single-molecule techniques. The stretched-out length of our DNA is enormous compared with the dimensions of a cell. To make DNA fit within a cell it is systematically wrapped around proteins (histone octamers), forming nucleosomes....

Single-molecule studies of the twisted, knotted, and broken genome

This thesis describes a series of single-molecule experiments aimed at understanding the physical properties of DNA itself and the proteins that interact with it. We developed and applied sensitive techniques that allowed us to directly probe the conformation and interactions of individual DNA molecules and proteins. Magnetic and optical...

Dynamic protein assemblies in homologous recombination with single DNA molecules

What happens when your DNA breaks? This thesis describes experimental work on the single-molecule level focusing on the interaction between DNA and DNA-repair proteins, in particular bacterial RecA and human Rad51, involved in homologous recombination. Homologous recombination and its central event of DNA strand exchange are essential processes...