Biology Reference
In-Depth Information
work with
E. coli
implicated the mismatch repair proteins MutL and MutS in
TCR and in mutation frequency decline (MFD),
13,14
although the reactions
reconstituted
in vitro
by Selby and Sancar did not require these proteins.
Although the RNA polymerase blocked at a lesion inhibits repair, it is an
essential participant in TCR. It plays an important role in scanning for damage
as it translocates, and having encountered a lesion, if blocked, it becomes a
target of
Mfd
. The interaction with the arrested RNA polymerase activates
Mfd
, which then recruits
UvrA
. The
subunit of the polymerase, coded by the
rpoB
gene, interacts with
Mfd
, and certain mutations affecting the N-terminal
region of the RpoB protein prevent this interaction and abolish TCR.
15-18
An unexpected observation was that in
E. coli
, a gene engineered to be
transcribed by the RNA polymerase of bacteriophage T7 showed TCR when it
was actively transcribed but not when it was downregulated.
19
Even more
surprising, the TCR promoted by the T7 polymerase
in vivo
was not observed
in an
mfd
strain, implicating
Mfd
in the response although Selby and Sancar
had shown that
Mfd
did not interact with the T7 polymerase
in vitro
.
12
The biochemical details of this phenomenon remain to be determined.
b
IV. The Role of Mfd in TCR
In 1956, Witkin
20
documented a phenomenon in UV-irradiated
E. coli
that
was later called ''mutation frequency decline'',
21
and she identified a gene (
mfd
)
associated with it.
2,22
The phenomenon was characterized by a decrease in
certain suppressor mutations when protein synthesis was inhibited in UV-
irradiated
E. coli
, and it did not occur in excision-deficient mutants or in
mfd
mutants. Subsequently, Bockrath and Palmer
23
presented evidence that MFD
reflected the repair of premutational lesions (probably T
CCPDs
24
)intRNA
genes and that the repair occurred more rapidly in the transcribed strand than in
the nontranscribed strand. They suggested that the nascent RNA transcript
might provide the template for efficient repair of the transcribed strand, a
plausible hypothesis at the time.
Based on the observations and ideas of Bockrath and Palmer, Selby and
Sancar cloned and sequenced the
mfd
gene, purified the
Mfd
protein, and
characterized it.
11,12,15,25,26
They ascertained that
Mfd
and TRCF were identi-
cal, and they established that when the RNA polymerase EC was blocked at a
lesion,
Mfd
could dissociate the polymerase and the nascent RNA transcript
from the DNA. Because part of the
Mfd
protein sequence was homologous to a
portion of the UvrB protein, and because they found that
Mfd
could bind
UvrA
(but not UvrB), they proposed that that
Mfd
could recruit
UvrA
, facilitating
repair. Subsequent studies have supported and extended these ideas, corrob-
orating the idea that the initiation of NER is different for TCR than for GGR.
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