Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK
(e-mail: j.svejstrup{at}cancer.org.uk)
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Summary |
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Key words: Transcription-coupled repair, Transcript elongation, Cockayne syndrome, Swi/Snf, Def1, Ubiquitylation, RNA polymerase II
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Introduction |
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Hanawalt and colleagues recognized almost two decades ago that DNA lesions
in the transcribed strand of an active gene are repaired much faster than
those in the non-transcribed strand or in the genome overall
(Bohr et al., 1985;
Mellon et al., 1986
;
Mellon et al., 1987
), but,
surprisingly, the molecular mechanism underlying such `transcription-coupled'
DNA repair (TCR) is still unclear. The importance of TCR is evident from the
fact that patients with deficiencies in the repair of lesions in the
transcribed strand of an active gene suffer from a very severe hereditary
disorder, Cockayne syndrome (CS) (de Boer
and Hoeijmakers, 2000
). Mutations in genes encoding basal
transcription factors or factors that are required for nucleotide excision
repair (NER) can give rise to both CS and xeroderma pigmentosum (XP),
including those in genes encoding two subunits of TFIIH (XPB and XPD), and XP
group G protein (XPG). By contrast, mutations in the genes encoding CS group A
(CSA) and CSB (yeast counterpart Rad26) can give rise to CS but do not result
in XP (Balajee and Bohr, 2000
;
Conaway and Conaway, 1999
;
de Boer and Hoeijmakers,
2000
). Common among all these factors is that they have been shown
to play a role in both transcription and DNA repair
(Feaver et al., 1993
;
Lee et al., 2002b
;
Lee et al., 2001
;
Lee et al., 2002c
;
Schaeffer et al., 1993
;
Selby and Sancar, 1997a
).
Interestingly, the repair deficiencies arising as a consequence of these
mutations do not underlie CS, since patients with the repair disorder
xeroderma pigmentosum are unable to repair UV-mediated DNA damage, both in the
transcribed and non-transcribed strand, yet do not have the same severe
clinical features that characterize patients who suffer (or also suffer) from
CS (de Boer and Hoeijmakers,
2000
; Lehmann,
2001
; Svejstrup,
2002b
). Symptoms of CS such as growth retardation, skeletal and
retinal abnormalities and progressive neural retardation are thus likely to be
caused by transcription deficiencies that may be related to DNA damage rather
than directly to failure to remove DNA damage from the genome. Very revealing
for the possible mechanism of TCR is the presence of ATPase motifs in the CSB
protein. These motifs are similar to those found in the Snf/Swi-family of
ATP-dependent chromatin-remodeling enzymes
(Eisen et al., 1995
).
In this Hypothesis, I discuss the results of four important recent
studies (Lee et al., 2002a;
Park et al., 2002
;
Saha et al., 2002
;
Woudstra et al., 2002
), which
are relevant to the mechanism of TCR in eukaryotes. On the basis of these
studies, I propose a model for the rescue of arrested Pol II complexes in
which RNA polymerases stalled at DNA lesions are not considered simply as
substrates for a subform of DNA repair (i.e. TCR) but rather as a problem for
transcription that can be solved in several fundamentally different ways. It
is, however, important to point out that TCR is turning out to be an extremely
complex phenomenon and that the model argued for here represents only one
current interpretation of often conflicting or at least confusing evidence.
For a more comprehensive review of the literature, the reader is referred to
recent reviews of the field (Balajee and
Bohr, 2000
; Conaway and
Conaway, 1999
; de Boer and
Hoeijmakers, 2000
; Svejstrup,
2002b
).
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A stalled polymerase triggers a rapid cellular response |
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The Swi/Snf-like ATPase activity of CSB/Rad26 and its role in removal of Pol II from DNA damage |
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Proteolysis of Pol II in response to DNA damage |
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Woudstra et al. purified yeast Rad26 from soluble (DNA-free) as well as
salt-stable chromatin and found that it exists in different forms. The form
isolated from the soluble fraction does not appear to be associated with any
other protein, whereas the form extracted and purified from chromatin is
associated with a novel protein, Def1. Cells lacking DEF1 have
phenotypes that indicate that this protein has a role in the DNA damage
response as well as in Pol II transcript elongation. Remarkably,
def1 cells are not UV sensitive and perform TCR efficiently.
However, when def1
is combined with mutations that reduce or
abolish the ability of cells to repair DNA damage, cells become extremely
sensitive to UV damage, which suggests that Def1 participates in a pathway
that represents an alternative to DNA repair. This pathway turns out to be the
ubiquitylation-mediated proteolysis pathway, and the target is Pol II. Thus,
cells lacking DEF1 are unable to ubiquitylate and degrade Pol II in
response to UV damage. Interestingly, cells lacking RAD26 behave in
the opposite way: here, Pol II is very rapidly and much more completely
destroyed in response to damage than is the case in wild-type cells. However,
in cells lacking both DEF1 and RAD26, Pol II degradation is
somewhat restored. Rad26 and Def1 might thus functionally interact in vivo to
regulate the degradation of Pol II in response to DNA damage
(Woudstra et al., 2002
).
Interestingly, work in mammalian cells has shown that ubiquitylation and Pol
II degradation are impaired, not increased, in the absence of functional CSB
(Bregman et al., 1996
;
Luo et al., 2001
;
McKay et al., 2001
). At first
glance, the function of yeast Rad26 and human CSB thus appears to differ
fundamentally. However, a possible explanation for the difference could be
that the function of both CSB and the presumed Def1 homologue is compromised
by mutation of CSB in man, whereas Rad26 and Def1 are able to perform their
functions independently in yeast. In support of this scenario, gel filtration
experiments have shown that (in contrast to yeast Rad26), human CSB normally
exists in a large protein complex (van
Gool et al., 1997
). The function of this entire complex (including
Pol II degradation) might thus be compromised by CSB mutation.
An important recent study by Sharp and colleagues supports the idea that
arrested Pol II is a target of ubiquitylation
(Lee et al., 2002a). Here, it
was shown that ubiquitylation of Pol II during transcription in vitro is
significantly induced by
-amanitin, which blocks Pol II transcript
elongation and also causes Pol II degradation inside cells
(Nguyen et al., 1996
). In
agreement with the above interpretation of the data from Woudstra et al.
(Woudstra et al., 2002
), Pol
II undergoes similar ubiquitylation on DNA containing cisplatin adducts (DNA
lesions) that arrest transcription (Lee et
al., 2002a
). Together, the data by Woudstra et al. and Lee et al.
thus suggest that Pol II is subject to ubiquitylation whenever it arrests
during transcription and that this can lead to its degradation
(Lee et al., 2002a
;
Woudstra et al., 2002
).
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A model for repair of damage-stalled Pol II elongation complexes |
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According to the model, if arrested Pol II complexes cannot be resolved by
general elongation factors or CSB/Rad26-related mechanisms, Pol II is now
ubiquitylated in a process that is at least partially dependent on Def1.
Whether there is damage or not, this clears the path for alternative pathways
of resolution. Because both repair and transcript elongation is affected by
proteolysis-independent functions of the 19S regulatory complex of the
proteasome (Ferdous et al.,
2001; Gillette et al.,
2001
; Russell et al.,
1999
), it is possible that ubiquitylation does not always result
in Pol II degradation. Johnston and co-workers have thus proposed that the 19S
complex might somehow manipulate the structure of the Pol II elongation
complex by proteolysis-independent mechanisms to facilitate transcription
(Ferdous et al., 2001
).
However, when all else fails, ubiquitylation of Pol II would eventually lead
to its degradation by the proteasome, allowing transcription of the gene to be
completed by the subsequent polymerase and repair to take place by
transcription-independent pathways.
It is important to remember that the action of CSB/Rad26 and ubiquitylation
factors such as Def1 is not confined to situations in which there is DNA
damage. These proteins are likely to have similar roles when cells try to
rescue Pol II elongation complexes that are stalled for reasons other than DNA
damage (such as DNA structure or sequence context, protein blocks, etc.). In
support of this idea, CSB affects transcript elongation of undamaged DNA in
vitro (Selby and Sancar,
1997a), and cells lacking RAD26 have transcription
defects in vivo (Lee et al.,
2001
). def1
cells also have defects consistent
with a role for this protein in transcript elongation in the absence of DNA
damage (Woudstra et al.,
2002
). The possibility that cells make overall transcription more
efficient by clearing Pol II `roadblocks' through proteolysis of the
polymerase in the absence of DNA damage clearly deserves further
attention.
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