Maarten C. Noom

Bacterial chromatin organization by H-NS protein unravelled using dual DNA manipulation

Both prokaryotic and eukaryotic organisms contain DNA bridging proteins, which can have regulatory or architectural functions. The molecular and mechanical details of such proteins are hard to obtain, in particular if they involve non-specific interactions. The bacterial nucleoid consists of hundreds of DNA loops, shaped in part by non-specific DNA bridging proteins such as histone-like nucleoid structuring protein (H-NS), leucine-responsive regulatory protein (Lrp) and SMC (structural maintenance of chromosomes) proteins. We have developed an optical tweezers instrument that can independently handle two DNA molecules, which allows the systematic investigation of protein-mediated DNA–DNA interactions. Here we use this technique to investigate the abundant non-specific nucleoid-associated protein H-NS, and show that H-NS is dynamically organized between two DNA molecules in register with their helical pitch. Our optical tweezers also allow us to carry out dynamic force spectroscopy on non-specific DNA binding proteins and thereby to determine an energy landscape for the H-NS–DNA interaction. Our results explain how the bacterial nucleoid can be effectively compacted and organized, but be dynamic in nature and accessible to DNA-tracking motor enzymes. Finally, our experimental approach is widely applicable to other DNA bridging proteins, as well as to complex DNA interactions involving multiple DNA molecules.

H-NS promotes looped domain formation in the bacterial chromosome

The bacterial chromosome is organized into loops, which constitute topologically isolated domains. It is unclear which proteins are responsible for the formation of the topological barriers between domains. The abundant DNA-binding histone-like nucleoid structuring protein (H-NS) is a key player in the organization and compaction of bacterial chromosomes [1,2]. The protein acts by bridging DNA duplexes [3], thus allowing for the formation of DNA loops. Here, genome-wide studies of H-NS binding suggest that this protein is directly involved in the formation or maintenance of topological domain barriers.

DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway

Type II restriction endonucleases protect bacteria against phage infections by cleaving recognition sites on foreign double-stranded DNA (dsDNA) with extraordinary specificity. This capability arises primarily from large conformational changes in enzyme and/or DNA upon target sequence recognition. In order to elucidate the connection between the mechanics and the chemistry of DNA recognition and cleavage, we used a single-molecule approach to measure rate changes in the reaction pathway of EcoRV and BamHI as a function of DNA tension. We show that the induced-fit rate of EcoRV is strongly reduced by such tension. In contrast, BamHI is found to be insensitive, providing evidence that both substrate binding and hydrolysis are not influenced by this force. Based on these results, we propose a mechanochemical model of induced-fit reactions on DNA, allowing determination of induced-fit rates and DNA bend angles. Finally, for both enzymes a strongly decreased association rate is obtained on stretched DNA, presumably due to the absence of intradomain dissociation/re-association between non-specific sites (jumping). The obtained results should apply to many other DNA-associated proteins.

Alba shapes the archaeal genome using a delicate balance of bridging and stiffening the DNA

Architectural proteins have an important role in shaping the genome and act as global regulators of gene expression. How these proteins jointly modulate genome plasticity is largely unknown. In archaea, one of the most abundant proteins, Alba, is considered to have a key role in organizing the genome. Here we characterize the multimodal architectural properties and interplay of the Alba1 and Alba2 proteins using single-molecule imaging and manipulation techniques. We demonstrate that the two paralogues can bridge and rigidify DNA and that the interplay between the two proteins influences the balance between these effects. Our data yield a structural model that explains the multimodal behaviour of Alba proteins and its impact on genome folding.

Protein-Mediated Molecular Bridging: A Key Mechanism in Biopolymer Organization

Protein-mediated bridging is ubiquitous and essential for shaping cellular structures in all organisms. Here we dissect this mechanism for a model system: the Histone-like Nucleoid-Structuring protein (H-NS). We present data from two complementary single-molecule assays that probe the H-NS-DNA interaction: a dynamic optical-trap-driven unzipping assay and an equilibrium H-NS-mediated DNA looping scanning force microscopy imaging assay. To quantitatively analyze and compare these assays, we employ what we consider a novel theoretical framework that describes the bridging motif. The interplay between the experiments and our theoretical model not only infers the effective interaction free energy, the bridging conformation and the duplex-duplex spacing, but also reveals a second, unresolved, cis-binding mode that challenges our current understanding of the role of bridging proteins in chromatin structure. We expect that this theoretical framework for describing protein-mediated bridging will be applicable to proteins acting in chromatin and cytoskeletal organization.

Visualizing the Formation and Collapse of DNA Toroids

In living organisms, DNA is generally confined into very small volumes. In most viruses, positively charged multivalent ions assist the condensation of DNA into tightly packed toroidal structures. Interestingly, such cations can also induce the spontaneous formation of DNA toroids in vitro. To resolve the condensation dynamics and stability of DNA toroids, we use a combination of optical tweezers and fluorescence imaging to visualize in real-time spermine-induced (de)condensation in single DNA molecules. By actively controlling the DNA extension, we are able to follow (de)condensation under tension with high temporal and spatial resolution. We show that both processes occur in a quantized manner, caused by individual DNA loops added onto or removed from a toroidal condensate that is much smaller than previously observed in similar experiments. Finally, we present an analytical model that qualitatively captures the experimentally observed features, including an apparent force plateau.

Unraveling DNA tori under tension

Motivated by recent experiments, we develop a model for DNA toroids under external tension. We find that tori are the equilibrium states for our model up to a critical tension, above which they become only metastable. Above this tension, we find a cascade of transitions between discrete toroid states that successively lower the winding number, until the ground state (rod) is reached. In this process, this model predicts a nearly constant force plateau as a function of extension, in agreement with experiment.

Archaeal DNA Organization: The Mechanism of Alba I & II Revealed

Throughout the kingdoms of life cells face a similar problem, namely the size of their genome is very large compared to the volume of the cell. Although each organism employs its own set of proteins to compact their genome, up to a factor of 10.000, the method of compaction seems highly conserved. Besides, DNA organization is known to be a key regulatory mechanism involved in many important processes such as gene regulation and DNA replication. The protein Alba, one of the most abundant proteins in Archaea, has been suggested to play an important role in DNA organization. Previous studies have shown that Alba binds as a dimer to non-specific DNA sequences and is able to condense DNA. However, little is known about its mechanism and structural role in DNA-organization. Here we show, using several single-molecule imaging and manipulation techniques such as AFM, double and quadruple optical tweezers, the condensing and possible regulation mechanism of Alba. From the surprising structural changes to the protein-bound DNA the binding constant, footprint and cooperativity factor are obtained by using a McGhee von Hippel analysis. We furthermore prove that the Archaea protein Alba II, which is thought to have a regulatory function, controls the binding efficiency of Alba I by changing the cooperativity of the Alba & Alba II protein mix.