Biology Reference
In-Depth Information
I. Structural Insights of Bacterial Nucleotide Excision Repair
A. Overview of the Process
Bacterial nucleotide excision repair (NER) is mediated by six proteins
1
(
Fig. 1
). Initial damage recognition is performed by UvrA, which acts as a
dimer. The cocrystal structure of a UvrA dimer bound to a DNA fragment
containing a fluorescein-modified thymine indicates that the damaged DNA fits
snugly into a channel formed by the UvrA dimer.
2
It is believed that positively
charged amino acids along this channel help to stabilize the binding of DNA to
UvrA.
2,3
Damage recognition is apparently mediated by the C-terminal zinc
fingers.
4
Most recently, it has been shown that these zinc fingers help to
communicate with the ATPase sites within the protein.
5
ATP binding promotes
UvrA dimerization, whereas ATP hydrolysis promotes monomerization. How-
ever, ATP hydrolysis is required for damage discrimination, as ATP-
g
-S abro-
gates damage recognition by increasing the nonspecific binding of UvrA.
6,7
These ATP-binding sites are also important for allowing damage verification
by UvrB,
5
as hydrolysis by UvrA is believed to promote UvrA release during
loading of UvrB at the site of damage.
8,9
UvrB has been determined to melt 4-6
bases by inserting a beta-hairpin through the two DNA strands containing the
DNA lesion.
10
Tyr96 at the base of the beta-hairpin is essential for damage
verification.
11,12
UvrB resembles monomeric helicases, and it is believed that
ATP hydrolysis of UvrB's helicase-like fold is essential for damage verification
and proper lesion positioning for binding and incision by UvrC.
8
UvrC contains two nuclease domains in the N-terminal and C-terminal
regions, required for dual incisions 3
0
and 5
0
on the damaged nucleotide, respec-
tively.
13,14
Once UvrC performs both endonucleolytic cuts, two additional pro-
teins, UvrD and DNA polymerase I (Pol I), are required to displace the
postincision complex, fill in the repair patch, and allow UvrB and UvrC to turn
over.
15
The final step in prokaryotic NER is the sealing of the remaining nick by
DNA ligase I.
B. Dynamics of the UvrA
2
B
2
-DNA Complex
Determination of the stoichiometry of the UvrAB complex has been contro-
versial, but two definitive experiments indicate that the working complex consists
of two subunits each of UvrA and UvrB.
16,17
There are three important problems
regarding the dynamics of the interaction of UvrA
2
B
2
with damagedDNA: (1) the
way the damage is handed off from the UvrA dimer to UvrB; (2) the nature of the
search mechanism; and (3) the rate-limiting step in damage processing by these
two proteins. Regarding the first question of damage handoff, one alternative is
that the damage site slides from the central location of the two zinc fingers at the
UvrA dimer interface to the beta-hairpin of UvrB.
2,18,19
Another more likely
