Biology Reference
In-Depth Information
beads, (ii) DNA, (iii) wash, and (iv) Qdot-bound proteins. TIRF is adapted
to illuminate the proteins through the lens by steering the excitation laser to
a subcritical angle; in practice, this simply requires moving the excitation
laser toward the center of the objective until the DNA tightropes can be
seen. Using this ''oblique angle fluorescence'' (OAF) configuration provides
considerable improvement in the signal to noise ratio over epi-fluorescence.
Visualization of specific proteins is mediated through tagging with quantum
dots (Qdots) (discussed later). Qdots are suitable for long acquisitions
because of their resistance to photobleaching while their extreme bright-
ness permits short integration times. Qdots also have very broad excitation
spectra permitting multicolor emissions to be recorded using just a single
excitation source. This latter point is extremely important as most DNA
enzymes work with partners to perform their job; such is the case for all
systems that exploit kinetic proofreading mechanisms. Moreover, as the
proteins are elevated far from any surface-bound Qdots, they do not
interfere with fluorescent spot discrimination. Finally, the density of
DNA can be tuned to high or low values. This permits a single protein on
a single DNA strand to be examined in the context of other closely juxta-
posed DNA molecules. This is close, but not a perfect equivalent, to
random coil DNA, where sequence-distant strands come within close
spatial proximity of the DNA-bound proteins, thereby making it possible
to study intersegmental transfer or even observe hopping directly
16
.
5. To image proteins working on DNA using any of the approaches above
first requires the conjugation of a fluorophore. Here we focus on strategies
for Qdot conjugation to allow single-molecule imaging during NER. In order
to watch UvrA and UvrB work together to search DNA, two different
strategies for conjugating Qdots were necessary
16,85
(see
Fig. 4
). Qdots are
commercially available with a number of different ligands attached to the
surface and a wide variety of approaches have been used to visualize single
molecules both
in vitro
and
in vivo.
86,87
The two most common are strepta-
vidin and antibodies. In order to conjugate UvrA to Qdots, a biotin ligase site
was engineered into the C terminus of UvrA. This 15-amino acid-sequence
contains a specific lysine that is covalently attached to biotin by biotin ligase.
This modification can be done during protein production within
E. coli
or
after purification using purified biotin ligase to levels approaching 95% of the
total protein. Mixing the biotinylated UvrA with excess Qdots ensures that
only one UvrA monomer is attached per Qdot. As multiple streptavidin
moieties are bound to the Qdot surface, atomic force microscopy is routinely
used to assure a 1:1 stoichiometry of attachment of protein to DNA.
16,85
Importantly, it is essential to ensure that the attachment of a Qdot does not
affect the function of the protein. Therefore, electrophoretic mobility shift
assays in agarose gels are used to ensure that the attachment of the Qdot to
