Biology Reference
In-Depth Information
III. Damage Searching by UvrA
2
and UvrA
2
B
2
The first aspect of any DNA protein interaction is specificity, which often
refers to the affinity of a protein for its cognate binding site. However, there is a
vast amount of very similar DNA, which leads to the question: How are targets
found? This is the central theme of this chapter and by using the single-
molecule techniques outlined earlier, we are able to begin to address this
question. It is likely that the NER system uses a combination of the many
possible mechanisms such as sliding, hopping, and intersegmental transfer.
Investigations at the single-molecule level have permitted direct observation
of these processes and also allow quantitative characterization of their relative
abundance. With more details emerging from such analyses, it may also become
clear why one mechanism is favored over another (
Fig. 5
).
Observation of UvrA
2
bound to DNA found it to remain statically bound for
7 s before detaching and binding elsewhere. This is exactly the equivalent of
a 3D search mechanism, where the DNA is searched through random encoun-
ters. However, it was noted that UvrA
2
appeared to jump to nearby strands,
with an average jump distance of 1.2
m
m.
16
The significance of this distance has
yet to be determined, but it may be a result of the architecture of the assay.
A high density of DNA tightropes resembles a sheet of binding surface; as a
molecule is released from the DNA, what is the probability that it will pass back
onto the sheet versus diffusing into solution? Does the relatively high incidence
of rebinding suggest that DNA keeps the protein close to itself until it finds the
right orientation to bind, possibly through electrostatics? If it is the latter, then
further studies using salt to shield these charges will provide valuable insight.
An approximation of the search time for a lesion suggested that the 3D mode of
search was very inefficient
16
; therefore, it is unlikely that UvrA
2
alone can
search the DNA effectively. Surprisingly, however, it was found that the addi-
tion of UvrB to form a UvrA
2
B
2
complex led to a change in the way UvrA
2
B
2
searched the DNA relative to UvrA
2
alone: UvrB collapsed the 3D search into
1D diffusion along the DNA.
16
Recalculation of the 1D search time resulted in
a huge increase in the rate at which DNA could be examined, bringing the
search time for the UvrA
2
B
2
molecules within the cells down from hours to
minutes. It is interesting to note that not all of the molecules participated in this
process; why is this so? One possibility is that the complex composition
(UvrA
2
B
2
vs. UvrA
2
B) may affect motility. It was found that the UvrAB com-
plexes used three different modes of motion: 1D diffusion, directed, and
paused. Directed motion was observed only in the presence of ATP; however,
it is possible that these represented under-sampled populations of diffusers.
Paused movers appeared to diffuse and then pause, often pausing at the same
site many times, and although the DNA was not intentionally damaged, some
nicks were detected. Perhaps these ''pausers'' were sensing damage but not
