Antoine van Oijen
Department of Biological Chemistry and Molecular Pharmacology
Harvard Medical School
Seeley G. Mudd Bldg. Room 204A
240 Longwood Ave., Boston, MA 02115
tel: (617) 432-5586; fax: (617) 738-0516
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
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.
J.B. Lee, R.K. Hite, S.M. Hamdan, X.S. Xie, C.C. Richardson, A.M. van
Oijen (2006) "DNA primase acts as a molecular brake in DNA replication,"
Nature 439: 621-624.
N.A. Tanner, S.M. Hamdan, J. Slobodan, K.V. Loscha, P.M. Schaeffer, N.E.
Dixon, A.M. van Oijen (2008) "Single-molecule studies of fork dynamics
in Escherichia coli DNA replication," Nature Structure &
Molecular Biology 15: 170-175.