Biology Reference
In-Depth Information
culture produced direct evidence that NER was not completely random with
respect to the DNA sequences repaired. For example, Zolan
et al.
3
observed
that furocoumarin adducts were repaired more slowly in the alpha DNA
sequences of African green monkey cells than in the bulk of the DNA. Similar
results were obtained for aflatoxin B1 adducts.
4
Shortly thereafter, Mansbridge
and Hanawalt
5
found that in cells derived from a patient with xeroderma
pigmentosum complementation group C (XPC), some domains of the DNA
were repaired rapidly after ultraviolet (UV) irradiation while the rest were
repaired slowly, if at all. Then Bohr
et al.
6
observed that in UV-irradiated
Chinese hamster ovary (CHO) cells, the active
DHFR
gene was repaired
more rapidly than the genome overall, and Mellon
et al.
7
showed that the
preferential repair of the
DHFR
gene in CHO and in human cells in culture
was due to the rapid repair of the transcribed strand of the expressed gene. The
nontranscribed strand was repaired at the same rate as the genome overall.
Extending those studies, the authors found that the transcribed strand of the
lac
operon of
E. coli
was repaired more rapidly than the nontranscribed strand
when the operon was induced, but not when it was repressed.
8
The studies
showing preferential repair of UV damage in transcriptionally active genes in
cultured CHO and human cells had led to the idea that chromatin might affect
the rate of repair, and that DNA damage in active genes might be more
accessible to repair complexes than damage in inactive regions of the ge-
nome.
6,9
The observation that the rapid repair was specific to the transcribed
strand of a gene made this idea unlikely, however,
7
especially because of the
results obtained with
E. coli
, which lacks the chromatin structure of eukaryotic
DNA. It then was proposed that RNA polymerase blocked at a lesion in the
transcribed strand might facilitate recognition of the damage by the NER
incision complex.
8
III. The Role of RNA Polymerase in TCR
Extending the analysis of the mechanism(s) underlying TCR, Selby and
Sancar used purified proteins
in vitro
to study the role of the
E. coli
RNA
polymerase in repair. They observed that a cyclobutane pyrimidine dimer
(CPD) in the transcribed strand of a gene blocked the RNA polymerase EC,
which then prevented incision of the DNA by the repair complex.
10
Thus, the
blocked RNA polymerase inhibited repair and could not directly account for
TCR. In further studies, Selby and Sancar isolated a protein that they called
''transcription repair coupling factor'' (TRCF). This protein was able to disso-
ciate the blocked RNA polymerase and the nascent RNA transcript from the
DNA and facilitate excision
in vitro
.
11
They later established that the
mfd
gene
of
E. coli
coded for this protein, also known as
Mfd
.
12
Curiously, subsequent
