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RESEARCH

Studying biological processes at the single-molecule level can offer us an improved understanding of the underlying molecular mechanisms. Through the removal of ensemble averaging, distributions and fluctuations of molecular properties can be characterized, transient intermediates identified, and catalytic mechanisms elucidated. Our group utilizes and further develops novel single-molecule techniques to study problems in the following fields:

DNA replication:

By combining the mechanical manipulation of individual DNA molecules with optical microscopy we are able to study the complex process of DNA replication at the single-molecule level. Using the bacteriophage T7 and E.coli replication machineries as model systems (in collaboration with Charles Richardson & Nick Dixon), we study how the different enzymatic activities at the replication fork (DNA unwinding, synthesis, priming) are orchestrated. In particular, we aim to understand how the continuous synthesis of nucleotides at the leading strand is coordinated to the discontinuous production of Okazaki fragments on the lagging strand, and how the priming of Okazaki fragments is regulated.

Further, we study the interrelationship between DNA replication and DNA repair in live E. coli cells (in collaboration with Myron Goodman, Roger Woodgate, and Mike Cox. In particular, we visualize individual, fluorescently tagged DNA polymerase in live E. coli cells and study how different repair polymerases are recruited to the genome upon the triggering of the SOS damage response.

Viral fusion:

Specific fusion of biological membranes is a central requirement for many cellular processes. It is the key molecular event during the entry of enveloped viruses into cells and represents an important target for antiviral therapeutics. Many structural and biochemical studies have contributed towards an understanding of the molecular workings of the viral proteins that mediate fusion, but little is known about the dynamics of the conformational changes and the nature of the potential cooperativity that are needed to catalyze the kinetically highly unfavorable fusion process. In collaboration with the vaccine-developing company Crucell and the group of Jolanda Smit, we are exploring these issues using a fundamentally new strategy: reconstituting viral fusion in vitro with only the bare minimum of molecular components and monitoring the dynamics of the fusion process at the single-particle level. These ‘molecular movies’ will allow us to dissect the reaction kinetics at a level of detail inaccessible to conventional ensemble experiments.

Membrane transporters:

In collaboration with Dirk-Jan Slotboom and Bert Poolman and the group of Arnold Driessen, we are using single-molecule fluorescence imaging techniques to understand the relationship between conformational dynamics of membrane transporters and their ability to transfer cargo across the cell membrane. In particular, we are interested in the SecYEG translocon and a variety of amino-acid and vitamine transporters.