Cell, Vol. 109, 823–834, June 28, 2002, Copyright 2002 by Cell Press
Requirement of Yeast RAD2, a Homolog of
Human XPG Gene, for Efficient RNA Polymerase II
Transcription: Implications for Cockayne Syndrome
additional protein factors not needed for the repair of
the nontranscribed DNA strand. The mechanism of TCR
has been elucidated in considerable detail in E. coli,
where the Mfd protein, which is a DNA-dependent ATP-
ase, displaces RNA polymerase from the lesion site in
a reaction requiring ATP hydrolysis. Subsequently, Mfd
recruits the excision nuclease to the lesion site (Selby
and Sancar, 1993). In humans, mutations in the Cock-
ayne syndrome (CS) genes, CSA and CSB, abolish TCR
of UV-damaged DNA (Venema et al., 1990; van Hoffen
et al., 1993). CSA, a WD repeat protein (Henning et al.,
a DNA-dependent ATPase (Selby and Sancar, 1997b),
could act in this process in a manner analogous to Mfd.
Because of a defect in the TCR of UV lesions, CS
individuals exhibit mild sun sensitivity; however, they
do not suffer from the high incidence of skin cancers
so characteristic of XP patients. Growth is severely re-
tarded in CS patients with the outward appearance of
cachetic dwarfism. In addition, CS individuals suffer
from impaired neurological development and mental re-
tardation. The mean age of death in CS is ?12 years
(Nance and Berry, 1992). Mutations in the CSA and CSB
genes account for ?90% of CS patients, and mutations
in the XPB, XPD, and XPG genes account for the re-
maining CS cases (Hanawalt, 2000).
The basis for the growth and neurological defects in
CS has remained unclear thus far. While the CS syn-
drome in individuals with mutations in the XPB and XPD
because of the lack of any evidence linking XPG to a
role in transcription, the transcriptional defect model
(Bootsma and Hoeijmakers, 1993; Qiu et al., 1993) has
been found inadequate to account for the association
of CS with mutations in the XPG gene. Recently, based
upon the observations that TCR of two oxidative lesions
(thymine glycol and 8-oxoguanine) requires CSB, XPB,
XPD, and XPG and that mutations in the XPB, XPD, and
XPG genes that cause CS also block the TCR of these
lesions, defective TCR of these oxidative lesions has
etal.,1997; LePageetal.,2000).Implicit inthisproposal
is the assumption that endogenous levels of thymine
glycol and 8-oxoguanine lesions reach such high levels
that they cause a significant inhibition of transcription,
which then causes the severe growth and develop-
mental defects of CS.
Since the lack of any evidence for the involvement of
XPG in transcription has been the biggest hurdle to the
the underlying cause of CS, here we utilize the yeast
S. cerevisiae to examine the role of RAD2, the yeast
counterpart of XPG, in transcription. The NER system
is highly conserved among eukaryotes from yeast to
humans, and a combination of Rad14, Rad4-Rad23,
RPA, TFIIH, Rad1-Rad10, and Rad2 proteins (which are
the respective counterparts of human XPA, XPC-
HR23B, RPA, TFIIH, XPF-ERCC1, and XPG) mediates
the dual incision of UV-damaged DNA, resulting in the
release of an ?30 nt lesion-containing DNA fragment.
Sung-Keun Lee,2Sung-Lim Yu,2Louise Prakash,
and Satya Prakash1
Sealy Center for Molecular Science
University of Texas Medical Branch
6.104 Blocker Medical Research Building
11th and Mechanic Streets
Galveston, Texas 77555
In addition to xeroderma pigmentosum, mutations in
drome (CS).Here, we provide evidencefor the involve-
ment of RAD2, the S. cerevisiae counterpart of XPG,
Inactivation of RAD26, the S. cerevisiae counterpart
of the human CSB gene, also causes a deficiency in
occurs in the absence of both the RAD2 and RAD26
genes. Growth is also retarded in the rad2? and
inhibition is seen in the rad2? rad26? double mutant.
cause of CS.
Nucleotide excision repair (NER) in eukaryotes is a com-
plex process requiring as many as 30 proteins. In hu-
mans, a defect in NER causes xeroderma pigmentosum
characterized by extreme sun sensitivity, abnormal pig-
larly in sun-exposed areas. In humans, XPA, XPC-
A (RPA) mediate the dual incision of UV-damaged DNA.
Of these proteins, XPA, XPC-HR23B, and RPA function
in the recognition of DNA lesions. TFIIH, an essential
RNA polymerase II (Pol II) transcription factor, contains
the two DNA helicases XPB and XPD, which act to open
the DNA helix during both transcription initiation and
NER. Following the unwinding of duplex DNA, the dam-
endonucleases XPG and ERCC1-XPF, which cleave this
strand on the 3? and the 5? sides of the lesion, respec-
tively, resulting in the release of an ?30 nucleotide frag-
ment containing the DNA lesion. The gap is subse-
quently filled in by replicative DNA synthesis (Sancar,
1996; Prakash and Prakash, 2000).
Even though UV lesions block transcription, UV dam-
age from the transcribed DNA strand is removed at a
faster rate than from the nontranscribed strand. This
phenomenon, known as transcription-coupled repair
(TCR; Mellon et al., 1987), requires the participation of
2Present address: WonKwang University, Iksan, Chonbuk 570-749,
Similar to their human counterparts, the yeast Rad14
step, and the Rad3 and Rad25 DNA helicases, present
in TFIIH and which are the yeast counterparts of human
XPD and XPB proteins, respectively, promote the open-
ing of the duplex DNA for dual incision by the Rad1-
Rad10 and Rad2 nucleases on the 5? and 3? sides of
the lesion, respectively (for review, see Sancar, 1996;
Prakash and Prakash, 2000). As in humans, the TCR of
UV-damaged DNA in S. cerevisiae requires, in addition
to the above-noted NER proteins, the Rad26 protein,
which is the yeast counterpart of human CSB protein
(van Gool et al., 1994), and like CSB, Rad26 is a DNA-
dependent ATPase (Guzder et al., 1996).
Purified CSB protein stimulates the rate of elongation
by RNA polymerase II (Pol II) on oligo(dC)-tailed DNA
template in the absence of other transcription factors
(Selby and Sancar, 1997a), and recently we have pro-
vided evidence for the involvement of the yeast RAD26
gene in Pol II-dependent transcription in vivo (Lee et al.,
2001). Here, we provide evidence for the involvement
of RAD2 in Pol II-dependent transcription. Interestingly,
we find that both transcription and growth are more
severely inhibited in the rad2? rad26? double mutant
than in the rad2? and rad26? single mutants. These
results indicate that RAD2 and RAD26 provide alternate
means for efficient transcription, and further, they impli-
cate transcriptional defects as the underlying cause of
growth impairment that occurs in the rad2?, rad26?,
and rad2? rad26? mutant strains under conditions that
would require the synthesis of new mRNAs. From these
studies, we infer that CS is likely a transcription syn-
drome and that growth and developmental defects in
CS could result from defects in transcription.
also examined GAL7 and GAL10 transcription in the
rad1? and the rad14? mutants, which, like the rad2?
mutant, are totally defective in NER (Prakash and Pra-
kash, 2000). Whereas GAL7 and GAL10 mRNAs were
as efficiently induced in the rad1? and rad14? strains
as in the wild-type strain, the levels of GAL10 mRNA
were lower in the rad2? and rad26? strains than in the
1). The levels of GAL7 mRNA were also reduced in the
rad2? and rad26? strains, but only at the earlier time
tion occurred in the rad2? rad26? double-mutant strain,
where GAL7 mRNA levels were greatly diminished and
GAL10 transcription was almost blocked (Figure 1). In
striking contrast, the rad1? rad26? and rad14? rad26?
strains exhibited the same levels and patterns of GAL
gene induction as the rad26? strain (data not shown).
The decline in transcription in the rad2? strain, thus, is
of NER-defective mutants.
Synergistic Enhancement of Growth Defects
in the rad2? rad26? Mutant
Although neither the rad2? or rad26? mutation has a
discernible effect on growth in rich YPD medium, we
found that growth of the rad2? rad26? double-mutant
strain is considerably retarded even in this medium (Fig-
ure 2A). We further investigated the growth characteris-
tics of the rad2?, rad26?, and rad2? rad26? mutant
cells by transferring them from glucose (YPD) to lactate
(YPL) medium at 30?C. Because adaptation to a new
carbon source would entail the synthesis of new pro-
teins, the growth impairment in these mutant strains
might become more apparent in YPL medium. While the
rad2? and rad26? mutant strains grow at a somewhat
slower rate in YPL medium than the wild-type strain, the
of growth upon transfer to this medium (Figure 2B). By
contrast, the rad1? and rad14? mutations had no ad-
verse effect on growth in YPD or YPL media (Figure
2), and when combined with the rad26? mutation, the
double mutants grew at the same rate as the rad26?
strain (data not shown). Thus, as for transcription, the
growth defects conferred upon yeast cells by the rad2?
mutation are specific to RAD2 and are not a general
feature of an NER defect.
Effect of rad2? Mutation on Galactose-Inducible
Synthesis of GAL7 and GAL10 mRNAs
Since CS patients are viable and the rad2? or rad26?
mutants display no obvious growth defects when yeast
cells are grown in rich nutritional conditions (YPD me-
dium), we expected the transcriptional defects in the
absence of RAD2 or RAD26 function to be subtle. To
be able to discern any such changes in transcription,
we examined the mRNA levels of genes in a state of
deficiency might become more apparent then. We also
reasoned that if the RAD2 and RAD26 genes provided
alternate means for efficient transcription, then a more
severe inhibition of transcription would occur upon the
simultaneous inactivation of both these genes.
To examine the inducible synthesis of GAL7 and
medium and mRNA levels were determined for the wild-
type, rad2?, rad26?, and rad2? rad26? strains. NER is
a very versatile DNA repair pathway, and in addition to
the removal of UV lesions, it functions in the removal of
abasic sites and a variety of other types of DNA lesions
(Huang et al., 1994; Reardon et al., 1997; Torres-Ramos
et al., 2000). To ascertain that any defect in transcription
function and not due to a general defect in NER, we
Inhibition of Transcription in 6AU-Treated
6-azauracil (6AU) inhibits enzymes in the nucleotide me-
tabolism pathway and leads to a depletion of the RNA
precursors GTP and UTP in yeast cells (Exinger and
Lacroute, 1992). Because of the nucleotide depletion,
Pol II’s elongation rate is slowed and it suffers more
arrest in 6AU-treated cells. Elongation factor TFIIS en-
ables Pol II to transcribe through intrinsic arrest sites in
DNA (Reines, 1994), and yeast cells lacking the DST1
gene, which encodes TFIIS, exhibit increased sensitivity
to 6AU (Nakanishi et al., 1995). Conditional mutations
in RPB2, the yeast gene encoding the second largest
subunit of Pol II, also confer a 6AU-sensitive phenotype
Requirement of RAD2 for Transcription
genome. This generates a duplication of the RAD2 gene in which
one copy is missing the 5? portion of the gene and is nonfunctional,
and the other copy is lacking only the C-terminal 209 amino acids
ofthe RAD2encodedprotein, andthisconstitutesthe rad2C?muta-
we used plasmid pR2.62, which is a Yip-based plasmid carrying the
yeast URA3 gene and containing the entire rad2 gene harboring the
E794A mutation, to transform the wild-type strain EMY74.7, which
is isogenic to EMY73 except that it contains the ura3-52 mutation.
Ura3?transformants, carrying both the wild-type RAD2 gene and
the mutant rad2 E794A gene, were grown on synthetic complete
(SC) medium lacking uracil. To obtain a strain that contained only
the rad2 E794A mutation, the Ura3?colonies were grown on plates
containing 5-fluoro-orotic acid (FOA). The rad2 E794A cells were
identified by UV sensitivity and the presence of only the mutant form
of the gene verified by Southern blotting.
Received: December 13, 2001
Revised: May 14, 2002
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