Biology Reference
In-Depth Information
unwinding of the helix to allow easy insertion of UvrB's beta-hairpin.
1
Still
unresolved is how the UvrA/UvrB interface is weakened to allow dissociation of
the UvrA dimer during loading of UvrB. One plausible explanation is that the
conformation of this interface is altered by damage-induced hydrolysis of ATP by
both UvrA and UvrB.
20
This review describes how single-molecule techniques
offer a new approach toward understanding these problems. In particular, we
focus on novel imaging platforms that have recently been developed to allow the
process of NER to be examined one molecule at a time without the influence of
flow or surfaces and to highlight the use of current protein-tagging technologies,
particularly quantum dots.
C. Kinetic Proofreading as Part of a Dynamic DNA
Damage Recognition Process: Role of ATP
As explained in the previous section, DNA damage recognition is a dynamic,
ATP-requiring process. Damage recognition, damage verification, and finally
DNA damage processing leading to incision, all require a high degree of specific-
ity, which is probably achieved by what has been termed ''kinetic proofreading''.
21-
23
It is postulated that to cope with the frequent small differences in binding
energy between target and nontarget sites during NER, high-energy protein-
substrate intermediate complexes are created using ATP; however, if the target is
not confirmed, the complex can dissociate. This process utilizes energy to generate
a time gap between initial protein binding and subsequent catalysis during which
the complex can dissociate and the catalysis (DNA incision) is aborted. By
including multiple proofreading steps prior to catalysis, damage specificity is
improved. For NER, we envision that once UvrA
2
senses damage, UvrA
2
utilizes
ATP hydrolysis to position UvrB for damage verification, representing the first
kinetic proofreading step. Following this, in the second kinetic proofreading step,
UvrB hydrolyses ATP and releases UvrA
2
, making reversal of this process improb-
able; this also helps to impose a specific conformation on the damaged DNA,
promoting efficient incision
11
(see
Fig. 1
). In this way, UvrC is probably ''captur-
ing'' this preincision UvrB-DNA complex during an ATPase cycle on UvrB.
11
This
occurs prior to the enzymatic commitment step, which in this case is the dual
incision of the DNA that alters the integrity of the genome.
21-23
II. So Few DNA Repair Proteins, So Much DNA: Defining
the Big Problem
A. Challenge of Repair Inside a Bacterial Cell
The average size of an
Escherichia coli
cell is about 1
m
m in diameter.
Packed inside this volume of
10
6
bp of genomic DNA,
making the base pair concentration in the millimolar range. An undamaged
10
15
L is 4.6
1
