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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:
 Prokaryotic DNA replication (in collaboration with Charles Richardson): 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 replication machinery as a model system, 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.
Eukaryotic replication (in collaboration with Johannes Walter): We are studying the activity of eukaryotic
replication proteins at the single-molecule level in Xenopus cell-free extract, an environment that
closely mimics the cellular context, but is still compatible with in vitro single-molecule techniques. We
use a combination of mechanical and optical single-molecule techniques to elucidate the mechanism
with which the putative eukaryotic helicase, the MCM2-7 complex, unwinds DNA. The use of single-molecule
techniques will shed light on the physical nature of the coupling between the helicase and the DNA polymerases
in the eukaryotic replication fork, an interaction that, when disrupted, triggers a cellular response to
DNA damage.
 Viral fusion (in collaboration with Steve Harrison): 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 molecular cooperativity among multiple copies of these proteins that may be needed to catalyze the kinetically highly unfavorable fusion process. In collaboration with Steve Harrison’s group (Department of BCMP, HMS), we are characterizing the role of cooperativity in viral membrane fusion by 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.
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