Until quite recently, chemical processes could only be investigated on a bulk level, which meant handling large numbers of molecules simultaneously resulting in the detection of averaged behavior of these molecules. The stresses and forces that molecules exert on each other in the course of reactions were not directly measurable. During the past few years, this situation has changed rapidly thanks to the development of methods for manipulating single molecules. Methods such as optical tweezers and scanning force microscopy (SFM) make it possible to follow in real-time and at a single molecule level, the movements, forces and strains that develop during the course of a reaction. They can also be used to exert external forces to influence reactions in the hope of discovering the rules that govern the inter-conversion of mechanical and chemical energy in these processes. This area of research includes biochemical processes as diverse as protein folding, DNA elasticity, protein-induced bending of DNA, the stress-induced catalysis of enzymes, and the behavior of molecular motors such as kinesin, myosin, and RNA polymerase.

Molecular Machines

The research in the group focuses on experimentally exploring the dynamic function of DNA enzymes and complex assemblies of such enzymes interacting with nucleic acids. Single molecule experiments using optical tweezers technology will be used for the exploration of the highly complex mechanical tasks of these enzymes of which many details are not yet understood.

	Many proteins work together in the DNA replication machinery. The 'mechanochemistry' of the DNA duplication process is highly complex and dynamic. DNA is unwound, actively separated, and the correct bases are incorporated while the whole machinery moves over the DNA swiftly.

The kind of machinery that controls for instance the structure, duplication, and transcription of DNA in cells is very complex and involves many regulatory pathways (see figure). The current central challenge in life sciences is trying to understand in detail how such machinery works. Elucidation of these processes will have far ranging implications for basic and applied science. The core processes have been intensively studied with biochemical methods and much is known about the static structure of many of the participating bio-molecules. However, the dynamic physical processes of the interaction of these bio-molecules are a very important part of the function of the machinery. Exploring these dynamics, often only possible with single-molecule experiments, is now possible and is a rich research field in biological physics for the future. These studies allows the closing of the gap between the structural studies and the functional assays; thus the static pictures of enzymes can now be supplemented with dynamic descriptions of their biological functioning. The increasingly microscopic and quantitative analysis methods will push the limits towards progressively more exact and physical/mathematical understanding of molecular functions. This goal is spanning boundaries between biology, biochemistry, physics, and mathematics.

Bacteriophage Phi 29

This project is centered around the study of an enzyme complex, bacteriophage Phi 29 head connector, which is speculated to be a rotary engine that uses the helicity of DNA to either pull DNA into viral capsids in the bacteriophage replication cycle, or to expel the DNA in the initial stages of infection. This project involves optical tweezers instrumentation to measure the forces exerted by the enzyme, and single molecule fluorescence techniques to test the hypothesis of a rotary engine.

	Bacteriophage Phi 29 Head Connector

Restriction Enzymes

In this project we study the dynamics of (type II) restriction enzymes. The biological function of these small bacterial proteins is to detect and destroy foreign DNA inside the cell body, enabling bacteria to defend themselves against viral attacks. For this defense mechanism restriction enzymes recognize and cut certain base pair sequences on double stranded DNA molecules with extraordinary specificity. Binding to a ‘recognition-site’ triggers a large conformational change in DNA and/or enzyme, permitting the cleavage of both strands.
Restriction enzymes are studied quite extensively with biochemical techniques and many crystal structures are known, however, the dynamics of the interaction between these enzymes and DNA is largely unresolved. In our lab we can catch and stretch single DNA molecules between beads held in optical tweezers. These DNA constructs are then used as templates for single molecule experiments on nonspecific and specific enzymes interaction with DNA. 

DNA compaction and transcription regulation

The chromosomal DNA in bacterial cells is organized and strongly compacted in order to confine it within the relatively small volume of these cells. A number of so-called nucleoid-associated proteins (e.g. HU, H-NS, IHF and Fis) are thought to be of essential importance for the dynamic organization and compaction of bacterial chromatin. In addition, these proteins play specific roles in various cellular processes, among which transcription. Regulation of transcription by nucleoid-associated proteins can take place at different stages of the transcription process and the number of mechanisms employed is therefore diverse. The aim of our studies is to gain insight into the dynamic aspects of the various mechanisms of transcription regulation employed by this type of proteins.

	H-NS specifically represses transcription at the rrnB P1 by trapping of RNA polymerase within a repression loop  (SFM image)

Collaborators

John Perona, University of California, Santa Barbara, CA, USA
Francesco Pavone, Lenz, Florence, IT
Rolf Wagner, Heinrich-Heine Universitat, Dusseldorf, D
Carlos Bustamante, University of California, Berkeley, CA, US
Mauro Modesti, Erasmus Universiteit, Rotterdam, NL

Key references

J. van Noort, S. Verbrugge, N. Goosen, C. Dekker, R.T. Dame
Dual architectural roles of HU: formation of flexible hinges and rigid filaments
Proc. Natl. Acad. Sci. USA 101 (2004) 6969-6974

R.T. Dame, G.J.L. Wuite
On the role of H-NS in the organization of bacterial chromatin. From bulk to single molecules and back
Biophys. J. 85 (2003) 4146-4148

G.J.L. Wuite, S.B. Smith, M. Young, D. Keller and C. Bustamante. 
Single Molecule Studies of the Effect of Template Tension on T7 DNA Polymerase Activity
Nature 404:103-106 (2000)

J. Davenport, G.J.L. Wuite, R. Landick, C. Bustamante
Single Molecule Study of Transcriptional Pausing and Arrest by E. coli RNA Polymerase
Science 287:2497-2500 (2000)