Biology Reference
In-Depth Information
E. coli
cell contains about 20, 50, and 20 copies of UvrA, UvrB, and UvrC
respectively, and their concentrations are approximately equivalent to 20, 50,
and 20 nM, respectively. During DNA damage-induced SOS induction, which
takes
20-40 min, the levels of UvrA and UvrB proteins may rise about 10-
fold, whereas the
uvrC
gene is not under SOS control. Assuming UvrC employs
the same search mechanism as UvrA and UvrB, it is likely that UvrC's search
process for the UvrB-DNA preincision complex would be rate-limiting for
NER. In addition to global NER, transcription-coupled repair occurs when
damage blocks the progression of transcribing RNA polymerase (RNAP).
24
The MFD or transcription-coupling protein, which contains a fold similar to
UvrB, helps recruit the repair machinery to the site of the damage-induced
stalled RNAP.
25
E. coli
K12 WT strain showed about 10% survival after 22 J/m
2
of 254 nmUV light (UVC),
26
and quantitative polymerase chain reaction (PCR)
analysis of the damaged
E. coli
indicated that UVC induced cyclobutane
pyrimdine dimers (CPD) and 6-4 photoproducts at a ratio of 2.1:1, respective-
ly.
27
A fluence of 100 J/m
2
of UVC produced about 0.4 photoproducts/kb, of
which about 16.5% and 36.7% of these lesions were repaired in 15 min on the
nontranscribed and transcribed strands of the
lacI
gene, respectively.
27,28
Thus,
assuming an initial noninduced state, some 10 UvrA
2
B
2
complexes were capa-
ble of processing about 1000 photoproducts in 15 min or about 6.6 lesions/
complex/min. If, however, there was ample induction of UvrA and UvrB pro-
teins, this rate of repair could be up to 10 times slower. Using a complementary
approach of monitoring removal of
4 CPD/pBR322 in
E. coli
, it was found
that the UvrABC system could remove about 0.045 dimers/plasmid (4.4 kb)/
min.
29
As pBR322 exists at about 10 copies per cell and
E. coli
has 4.6
10
6
bp
genome, also irradiated in this experiment, that is equivalent to a total of about
45 CPD/min being processed by roughly 10 UvrA
2
B
2
complexes or about
4.5 CPD/complex/min. Together these conditions give a repair rate of 1 pho-
toproduct/1-2 kbp in roughly 10-100 s/UvrA
2
B
2
complex. How can such a
small number of proteins search through a vast sea of undamaged DNA to
quickly repair that quantity of DNA damage? This fundamental problem in
NER is discussed in more detail in the following section.
B. Potential Modes of Damage Site Location
The question of how proteins find their cognate binding sites in the
presence of excess DNA has been of great interest for over 50 years and several
search mechanisms have been proposed (reviewed in Ref.
30
). Despite the
large number of proteins packed into the small volume of a bacterial cell,
proteins diffuse rapidly. The green fluorescence protein (GFP), MW of
30 kDa, was found to have a three-dimensional (3D) diffusion constant of
about 7.7
m
m
2
/s.
31
This rapid Brownian motion is caused by collisions with
water molecules such that a single GFP molecule can diffuse the width of a
