Biology Reference
In-Depth Information
addition, as it has an affinity for UvrB and shares a binding site with UvrA on
UvrB, it seems that a useful switch has evolved, preventing interaction with a
searching complex until a lesion is found. However, UvrB is in excess of UvrA
and therefore it is possible that UvrB interacts with UvrC,
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sequestering it
from interacting with DNA until a preincision complex is found. Therefore,
perhaps, UvrB forms a complex with UvrC and accompanies it to the preinci-
sion complex and at the same time protects DNA from spurious incisions.
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Single-molecule approaches offer another advantage beyond the physical
description of lesion search: direct observation of repair in action being made
possible, revealing both the sequence of events and their kinetics. The canon-
ical mechanism of NER involves a complex of UvrA and UvrB searching for
lesions, followed by UvrA's ejection upon encountering damage. By observing
this process, it will be possible to confirm this sequence of events and also
determine the kinetics of UvrA release once the lesion is encountered. Fur-
thermore, abortive complexes formed stochastically
99,102
that may contribute
to kinetic proofreading will also be detected. Together these data will provide
a comprehensive understanding of how the preincision complex is formed.
The next process of UvrC recruitment can also be studied similarly and, as
DNA helicase working together with DNA polymerase I has been proposed to
remove UvrC and the damaged oligonucleotide,
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we will also be able
to discover whether UvrB is associated with the damaged strand or bound to
the opposite strand, and whether UvrB is present as a dimer or a monomer.
This latter question links back to our earlier discussion of hopping versus
sliding; one of the key arguments against sliding is that it probes only one
strand. Therefore, is the role of the UvrA
2
B
2
complex to permit UvrB loading
onto either strand? All these questions can be studied using multicolor direct
imaging of repair proteins labeled with differently colored Qdots.
The physical principles that underlie NER are critical to our understanding
of this process. Therefore, it is important that bulk and single-molecule studies
go hand in hand. Furthermore, investigations in simple bacterial systems are an
ideal platform for understanding these new concepts; diving into more complex
mammalian counterpart systems will preclude a thorough understanding.
Therefore, it is imperative that we revisit the bacterial system to achieve a
robust grounding in the principles that likely underlie mammalian NER.
IV. Future Directions
A. Observing Protein Nanomachines at Work
The end goal of the investigations of protein machines working on DNA is
to provide a kinetically correct, ordered mechanism. NER offers an ideal
platform for creating such a view of DNA repair; it is a relatively simple
