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Quantifying the physics inside the genome one molecule at a time
The genetic information of an organism is encoded in the base pair
sequence of its DNA. Many specialized proteins are involved in
organizing, preserving and processing the vast amounts of information
on the DNA. In order to do this swiftly and correctly these proteins
have to move quickly and accurately along and/or around the DNA
constantly rearranging it. In order to elucidate these kind of
processes we perform single-molecule experiments on model systems such
as restriction enzymes, DNA polymerases and repair proteins. The data
we use to extract forces, energies and mechanochemistry driving these
dynamic transactions. The results obtained from these model systems
are then generalized and thought to be applicable to many DNA-protein
interactions. In particular, I will report experiments that use a
combination of fluorescence microscopy and optical tweezers to :
(i) directly visualize the DNA overstretching transition and
demonstrate that its origin is the cooperative melting of the two DNA
strands. In the experiments we use intercalating dyes and
fluorescently labeled single-stranded binding proteins to specifically
visualize double- and single-stranded segments in DNA molecules
undergoing the transition. Our data unambiguously show that the
overstretching transition comprises a gradual conversion from
double-stranded to single-stranded DNA, in agreement with the
force-induced melting model.
(ii) image single Rad51 monomers disassembling from nucleoprotein
filaments formed on double-stranded DNA molecules. We quantified the
fluorescence of disassembling nucleoprotein patches and found that
disassembly occurs in bursts interspersed by long pauses. By applying
external tension to these filaments, we find that disassembly slows
down and can even be stalled. Upon relaxation of a stalled complex,
pauses were suppressed resulting in a large burst. These results imply
that tension-dependent Rad51 disassembly takes place only from
filament ends, after tension-independent ATP hydrolysis.
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