Nucleotide Excision Repair, Genome Stability, and Human Disease: New Insight from Model Systems
ABSTRACT Nucleotide excision repair (NER) is one of several DNA repair pathways that are universal throughout phylogeny. NER has a broad substrate specificity and is capable of removing several classes of lesions to the DNA, including those that accumulate upon exposure to UV radiation. The loss of this activity in NER-defective mutants gives rise to characteristic sensitivities to UV that, in humans, is manifested as a greatly elevated sensitivity to exposure to the sun. Xeroderma pigmentosum (XP), Cockaynes syndrome (CS), and trichothiodystrophy (TTD) are three, rare, recessively inherited human diseases that are linked to these defects. Interestingly, some of the symptoms in afflicted individuals appear to be due to defects in transcription, the result of the dual functionality of several components of the NER apparatus as parts of transcription factor IIH (TFIIH). Studies with several model systems have revealed that the genetic and biochemical features of NER are extraordinarily conserved in eukaryotes. One system that has been studied very closely is the budding yeast Saccharomyces cerevisiae . While many yeast NER mutants display the expected increases in UV sensitivity and defective transcription, other interesting phenotypes have also been observed. Elevated mutation and recombination rates, as well as increased frequencies of genome rearrangement by retrotransposon movement and recombination between short genomic sequences have been documented. The potential relevance of these novel phenotypes to disease in humans is discussed.
- SourceAvailable from: Adam M Bailis[Show abstract] [Hide abstract]
ABSTRACT: Increased mitotic recombination enhances the risk for loss of heterozygosity, which contributes to the generation of cancer in humans. Defective DNA replication can result in elevated levels of recombination as well as mutagenesis and chromosome loss. In the yeast Saccharomyces cerevisiae, a null allele of the RAD27 gene, which encodes a structure-specific nuclease involved in Okazaki fragment processing, stimulates mutation and homologous recombination. Similarly, rad3-102, an allele of the gene RAD3, which encodes an essential helicase subunit of the core TFIIH transcription initiation and DNA repairosome complexes confers a hyper-recombinagenic and hypermutagenic phenotype. Combining the rad27 null allele with rad3-102 dramatically stimulated interhomolog recombination and chromosome loss but did not affect unequal sister-chromatid recombination, direct-repeat recombination, or mutation. Interestingly, the percentage of cells with Rad52-YFP foci also increased in the double-mutant haploids, suggesting that rad3-102 may increase lesions that elicit a response by the recombination machinery or, alternatively, stabilize recombinagenic lesions generated by DNA replication failure. This net increase in lesions led to a synthetic growth defect in haploids that is relieved in diploids, consistent with rad3-102 stimulating the generation and rescue of collapsed replication forks by recombination between homologs.Genetics 08/2007; 176(3):1391-402. · 4.39 Impact Factor
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ABSTRACT: The transcription and DNA repair factor TFIIH is composed of 10 subunits. Mutations in the XPB, XPD, and p8 subunits are genetically linked to human diseases, including cancer. However, no reports of mutations in other TFIIH subunits have been reported in higher eukaryotes. Here, we analyze at genetic, molecular, and biochemical levels the Drosophila melanogaster p52 (DMP52) subunit of TFIIH. We found that DMP52 is encoded by the gene marionette in Drosophila and that a defective DMP52 produces UV light-sensitive flies and specific phenotypes during development: organisms are smaller than their wild-type siblings and present tumors and chromosomal instability. The human homologue of DMP52 partially rescues some of these phenotypes. Some of the defects observed in the fly caused by mutations in DMP52 generate trichothiodystrophy and cancer-like phenotypes. Biochemical analysis of DMP52 point mutations introduced in human p52 at positions homologous to those of defects in DMP52 destabilize the interaction between p52 and XPB, another TFIIH subunit, thus compromising the assembly of the complex. This study significantly extends the role of p52 in regulating XPB ATPase activity and, consequently, both its transcriptional and nucleotide excision repair functions.Molecular and Cellular Biology 06/2007; 27(10):3640-50. · 5.37 Impact Factor
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ABSTRACT: Excision repair cross-complementation group 4 gene (ERCC4/XPF) plays an important role in nucleotide excision repair and participates in removal of DNA interstrand cross-links and DNA double-strand breaks. Single nucleotide polymorphisms (SNPs) in ERCC4 may impact repair capacity and affect cancer susceptibility. In this case-control study, we evaluated associations of four selected potentially functional SNPs in ERCC4 with risk of squamous cell carcinoma of the head and neck (SCCHN) in 1,040 non-Hispanic white patients with SCCHN and 1,046 cancer-free matched controls. We found that the variant GG genotype of rs2276466 was significantly associated with a decreased risk of SCCHN (OR = 0.69, 95% CI 0.50-0.96), and that the variant TT genotype of rs3136038 showed a borderline significant decreased risk with SCCHN (OR = 0.76, 95% CI: 0.58-1.01) in the recessive model. Such protective effects were more evident in oropharyngeal cancer (OR = 0.61, 95% CI: 0.40-0.92 for rs2276466; OR = 0.69, 95% CI: 0.48-0.98 for rs3136038). No significant associations were found for the other two SNPs (rs1800067 and rs1799798). In addition, individuals with the rs2276466 GG or with the rs3136038 TT genotypes had higher levels of ERCC4 mRNA expression than those with the corresponding wild-type genotypes in 90 Epstein-Barr virus-transformed lymphoblastoid cell lines derived from Caucasians. These results suggest that these two SNPs (rs2276466 and rs3136038) in ERCC4 may be functional and contribute to SCCHN susceptibility. However, our findings need to be replicated in further large epidemiological and functional studies.PLoS ONE 01/2012; 7(7):e41853. · 3.53 Impact Factor
© 2002 Hindawi Publishing Corporation
Journal of Biomedicine and Biotechnology • 2:2 (2002) 1–6 • PII. S1110724302201023 • http://jbb.hindawi.com
Nucleotide Excision Repair, Genome Stability, and
Human Disease: New Insight from Model Systems
David J. Garfinkel1and Adam M. Bailis2∗
1Gene Regulation and Chromosome Biology Laboratory, NCI at Frederick, Frederick, MD 21702, USA
2Division of Molecular Biology, Beckman Research Institute of the City of Hope,
City of Hope National Medical Center, Duarte, CA 91010, USA
Revised 26 November 2001; Accepted 11 December 2001
Nucleotide excision repair (NER) is one of several DNA repair pathways that are universal throughout phylogeny. NER has a broad
to UV radiation. The loss of this activity in NER-defective mutants gives rise to characteristic sensitivities to UV that, in humans, is
manifested as a greatly elevated sensitivity to exposure to the sun. Xeroderma pigmentosum (XP), Cockaynes syndrome (CS), and
trichothiodystrophy (TTD) are three, rare, recessively inherited human diseases that are linked to these defects. Interestingly, some
of the symptoms in afflicted individuals appear to be due to defects in transcription, the result of the dual functionality of several
components of the NER apparatus as parts of transcription factor IIH (TFIIH). Studies with several model systems have revealed
that the genetic and biochemical features of NER are extraordinarily conserved in eukaryotes. One system that has been studied very
and defective transcription, other interesting phenotypes have also been observed. Elevated mutation and recombination rates, as
well as increased frequencies of genome rearrangement by retrotransposon movement and recombination between short genomic
sequences have been documented. The potential relevance of these novel phenotypes to disease in humans is discussed.
DNA REPAIR AND DISEASE
The maintenance of the integrity of DNA is of a para-
itory for all genetic information. A network of biochemical
pathways exists in all cells to maintain the informational and
in part, by the types of modifications to the DNA to which
they respond. The dissection of these pathways has involved
a coordinated biochemical, genetic, and molecular biologi-
cal approach where specific enzymatic defects are correlated
with mutations at particular chromosomal loci, and distinc-
tive cellular phenotypes. In humans, the specific DNA repair
defects have been correlated with particular inherited dis-
eases. Hereditary nonpolyposis colorectal cancer, for exam-
ple, is a disorder associated with mutations in genes encod-
ing components of the mismatch repair apparatus that lead
in genome instability . Similarly, a trio of phenotypically
disparate diseases, XP, CS, and TTD have been connected
with mutations in genes that encode subunits of the NER
and TFIIH apparatus, that confer measurable defects in NER
diseases most likely reflects the multiple biochemical defects
caused by the mutations, some of which have not been ex-
tensively explored. This work focus on some of the less de-
scribed effects of mutations in the NER/TFIIH apparatus
on genome stability, and their potential implications with re-
spect to human disease.
NUCLEOTIDE EXCISION REPAIR
NER plays a critical role in the maintenance of genomic
integrity because of its broad substrate specifity. It plays an
important role in the removal of such diverse lesions as UV
light induced photoproducts as well as chemically induced
bulky adducts, crosslinks, and oxidized bases. NER has been
functionally conserved throughout phylogeny, although the
apparatus in prokaryotes and eukaryotes is substantially dif-
to 30 proteins and has been reconstituted in vitro[5, 6].NER
occurs in the context of two distinct pathways related to the
way the lesions are identified. General genome repair (GGR)
ognized by the XPC/HHR23B complex . Transcription
coupled repair (TCR) is focused exclusively on the repair of
II stalled at a lesion by the CSA and CSB proteins [9, 10].The
XPA protein then orchestrates the assembly of a complex in-
cluding the single-stranded DNA binding protein RPA and
the core of the TFIIH complex at the site of the lesion .
ATP-dependent process that involves the helicase activities of
2D. J. Garfinkel and A. M. Bailis2:2 (2002)
the XPB and XPD proteins, components of the TFIIH core
complex [12, 13]. Interestingly, only the helicase activity of
XPB is required for TFIIH-mediated melting of promoter
DNA during the initiation of transcription, while XPD he-
licase activity plays a minor role in RNA polymerase II pro-
moter escape . Two structure-specific endonucleases, the
ERCC1/XPF heterodimer, that cleaves on the 3’ side of the
lesion,andXPG,thatcleavesonthe 5’ sideofthe lesion,are
responsible for the removal of a 24–32 base oligonucleotide
containing the lesion [15, 16]. The resulting gap is filled by
DNA polymerases δ and ε, and repair is completed by liga-
NER PHOTOSENSITIVITY SYNDROMES
XP, CS, and TTD have been ascribed to changes in NER
and TFIIH function. Complementation analysis, conducted
by analyzing the phenotypes of cells derived from the fusion
of cells from different patients, identified several comple-
tified for XP. Two complementation groups, CS-A and CS-B,
have been identified for CS. Three complementation groups,
XP-B, XP-D, and XP-G, have been determined for the com-
bined CS/XP patients. Three complementation groups, XP-
B, XP-D, and TTD-A, have been identified for TTD. Most of
the genes corresponding to these complementation groups
have been cloned and the mutations responsible for disease
in individual kindreds have been identified.
XP, CS, and TTD patients have distinct symptoms that
likely reflect the participation of the mutant proteins in mul-
tiple biochemical processes. In patients, symptoms can often
be correlated with specific biochemical defects. An example
terations of the texture and pigmentation of the skin that are
. CS patients present symptoms of poor growth and de-
velopmental deformity that are most likely due to defec-
tive transcription. Accordingly, extracts of cells from patients
with particular CSA and CSB mutations have been shown to
support reduced levels of RNA polymerase II mediated tran-
scription, as have cells from patients with combined XP/CS
disease that are due to a mutation in XPB . TTD patients
suffer from brittle hair and nails, scaly skin and developmen-
tal abnormalities that are probably related to defects in NER
was correlated with temperature sensitive transcription and
STUDIES WITH MODEL SYSTEMS
The extraordinary level of conservation of the NER and
TFIIH apparatus among eukaryotes permits the extensive
use of model systems to better explore the genetic and bio-
chemical control of theses processes. Studies in rodents, flies,
plants, and fungi have all contributed to the understanding
of the role played by the NER and TFIIH apparatus in eu-
karyotes [21, 22, 23, 24, 25, 26]. However, trangenic mice
and budding yeast have been particularly helpful in study-
ing NER and TFIIH at the molecular level, as well as their
effects on the phenotype of the organism. For example, in a
leles of XPA or CSB were found to be extremely sensitive to a
carcinogen that forms bulky adducts on DNA, while an XPC
null strain was not . This suggests that XPA-dependent
and CSB-dependent TCR is critical for survival to exposure
to an important class of carcinogens, while XPC-dependent
GGR is not. Such studies are very important in determining
how mammals respond to particular types of DNA damage.
Mice have also been used to further explore the phenotypic
effects of NER mutations known to cause disease in humans.
gous for an XPD mutation analogous to the one found in
several NER-deficient TTD patients . The mice exhib-
ited hypersensitivity to UV exposure and a cellular NER de-
fect that was very similar to the TTD symptomology but,
also exhibited a marked propensity toward UV-induced and
carcinogen-induced skin cancer that was not observed in pa-
tients. This suggests the possibility that the NER defects ob-
served in TTD patients could lead to cancer, an observation
of potential clinical importance.
malian cells has been essentially contemporaneous. The ob-
servation that NER and transcription are genetically and
biochemically linked occurred nearly simultaneously in the
two systems, significantly accelerating the description of eu-
karyotic NER and transcription initiation at the molecular
level [29, 30]. The degree of similarity between the appa-
ratus in humans and yeast is extraordinary, permitting in-
vestigators to examine the impact of disease causing alleles
of human NER genes on yeast. In one study, two TTD al-
leles of XPD were unable to complement the lethal effect
of a null allele of the RAD3 gene, the budding yeast ho-
molog of XPD, while wild-type and helicase-defective alle-
les of XPD were able to complement . Since the essential
function of Rad3 /XPD is thought to be its role in tran-
les confer a transcription defect, and, therefore, that the dis-
ease may be due to defective transcription of a critical gene,
NER, TFIIH AND THE MAINTENANCE OF GENOME
STABILITY IN BUDDING YEAST
The careful study of NER in budding yeast has revealed
its impact on cellular processes yet to be recognized in other
sight into clinically important roles for the human NER ap-
the disease phenotype is complex as in CS and TTD. An im-
portant example is provided by the RAD1 and RAD10 genes,
2:2 (2002) Nucleotide Excision Repair, Genome Stability, and Human Disease. New insight from model systems3
IIaII IIIIVV VI
3551 69 88225239455468 533554587 613 654671
240457 470535 556589 615 656673
Figure 1. Amino acid changes in the human XPD and budding yeast Rad3 helicases that lead to phenotypic changes. The primary amino acid sequences of
human XPD and yeast Rad3 are represented by the black bar. The seven conserved helicase domains are represented by white boxes and are named according
to the accepted nomenclature . The positions of these domains in the primary sequence are listed in bold print as described in . Positions of altered
residues in the primary sequence of XPD in XP, XP/CS, and TTD patients  are marked with a light hash mark above the black bar. The corresponding
diseases are marked with an “X” for XP, a “C” for XP/CS, and a “T” for TTD. The amino acid changes are listed in parentheses. Changes in the Rad3 primary
sequence [38, 39] are represented by hash marks below the black bar. The corresponding phenotypic changes are denoted by a “U” for UV sensitive, an “R”
for elevated recombination, and/or mutation, and a “T” for transcription defective. Corresponding amino acid changes are listed in parentheses.
homologs of XPF and ERCC1. Null mutations in RAD1
of homologous recombination [32, 33]. Studies analyzing
the recombinational repair of defined double-strand breaks
(DSBs) revealed that RAD1 and RAD10 were required to re-
move nonhomologous sequences from the ends of recom-
bining molecules, an important step in the creation of cer-
a critical role in recombination between short sequences,
which is likely to be vital for the maintenance of genome sta-
bility . All of these effects are apt to be related to the role
of the Rad1-Rad10 nuclease in the cleavage of important re-
combination intermediates [34, 35, 36].
The TFIIH machinery has also been implicated in the
maintenance of genome stability. Mutant alleles of RAD3,
SSL1, and SSL2 (homologs of XPD, P44, and XPB) have
been isolated that confer elevated rates of mutation and
recombination. Several rad3
that exhibit increased rates of mutation and/or recombi-
nation but not defective transcription nor NER, indicat-
ing that Rad3 possesses important cellular activities that are
mutants have been isolated8
independent of transcription and NER [37, 38]. Interest-
ingly, a pair of these mutant alleles, rad3-101 (S74F) and
rad3-102 (H661Y), accumulate DSBs and are synthetically
lethal in combination with mutations in recombinational
repair genes, strongly suggesting that Rad3 influences ei-
ther the creation of DSBs, or their processing by homol-
ogous recombination . Another allele of RAD3, rad3-
G595R, blocks the degradation of DSBs, which stimulates
genome rearrangement by recombination between short re-
peated sequences [35, 36, 37, 38, 39, 40, 41]. The anal-
ogous mutations in XPD are of potential medical impor-
tance as they would be within regions that encode con-
served helicase domains of the protein that are mutated in
XP, TTD, and XP/CS patients (Figure 1), [42, 43, 44]. Since
specific mutations in SSL1 and SSL2 that encode additional
members of the heteromeric core of budding yeast TFIIH
 also stimulate short-sequence recombination (SSR) by
blocking the degradation of DSBs [40, 41], it appears that
TFIIH acts to maintain genome stability by restricting re-
combination between repetitive DNA sequences. We specu-
late that SSR could potentially contribute to the symptoms
4 D. J. Garfinkel and A. M. Bailis2:2 (2002)
observed in XP, XP-CS, and TTD patients, as SSR in the
human genome has been found to lead to a variety of dis-
of the retrotransposon Ty1, TFIIH plays an additional, crit-
ical role in the maintenance of genome stability in budding
yeast. Ty1 can move either by integrase mediated transpo-
sition, or by insertion of Ty cDNA into existing Ty or δ ele-
bination . Components of TFIIH were discovered to play
a role in restricting Ty mobility when specific rad3 and ssl2
. Other rad3 and ssl2 mutants that confer extreme UV
sensitivity do not stimulate Ty mobility, separating the NER
and Ty maintenance functions of these genes. The increases
in Ty mobility were correlated with a substantial elevation of
the steady-state level of Ty1 cDNA, suggesting either that the
rad3- rtt (regulator of Ty transposition) and ssl2- rtt mu-
tations increase the synthesis of Ty1 cDNA, or decrease its
degradation. Importantly, the level of Ty1 RNA and Ty1 pro-
teins remained unchanged in the rad3 and ssl2 mutants. An-
other interesting observation is that other NER genes, such
as RAD2 and RAD1 do not appear to be involved in Ty trans-
In a subsequent study, the increased level of Ty1 cDNA
was found not to be due to increased cDNA synthesis sug-
gesting that TFIIH plays a role in the degradation of Ty1
cDNA . This same study linked TFIIH-control of SSR
and Ty1 movement by showing that rad3-G595R and ssl2-
rtt increase both SSR and Ty1 movement while increasing
the stability of both Ty1 cDNA and DSBs. It remains unclear
whether TFIIH plays a direct or an indirect role in the degra-
dation of the ends of DNA molecules, however, the helicase
activities of Rad3 and Ssl2 suggest that TFIIH could play a
role in opening the ends of DNA molecules, thereby facili-
tating their processing by exonucleases.
Blocking the degradation of DSBs and Ty1 cDNAs could
enhance Ty1 movement and SSR in multiple ways. It could
preserve sequences at the ends of the molecules that are im-
portant for SSR and Ty1 movement. For example, a DSB in
a short repetitive sequence creates ends that are never far
from the border of homology with potential donor repeats,
their ability to pair and recombine. Similarly, loss of the se-
quences at the ends of Ty1 cDNA would block transposition
by Ty1 integrase. Another way (that increasing the stability
of DNA ends might elevate SSR and Ty mobility) could be
by prolonging the signal that elicits the DNA damage check-
point. Pausing the cell cycle for a protracted period may im-
prove the likelihood that the ends of DSBs in short repeats
can find homologous sequences, or that the Ty1 preintegra-
tion complex can complete a transposition event. Changes
in checkpoint activity have been shown to affect the fre-
quencies of other genome rearrangements . These and
other mechanisms could be working simultaneously to in-
crease genome rearrangement in rad3 and ssl2 mutant bud-
ding yeast. By extension, similar defects in the cells of XP,
XP-CS, or TTD patients with mutations in the XPD or XPB
genes might contribute to these diseases.
This research was sponsored in part by the National Can-
cer Institute, Department of Health and Human Services.
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the RAD52 epistasis group inhibit Ty1 transposition.
 Fasullo M, Bennett T, AhChing P, Koudelik J. The
Saccharomyces cerevisiae RAD9 checkpoint reduces
the DNA damage-associated stimulation of directed
translocations. Mol Cell Biol. 1998;18(3):1190–1200.
2Please note that genes and enzymes symbols have to be ital-
icized, while protein symbols are roman, so you are kindly
to ensure that they are correct throughout.
3 The highlighted 3?and 5?mean the third and the fifth?
4We added “have been” .
5Please indicate if “Rad3” is a gene name or not.
6In the legend of Figure 1, we changed “as previously described
” to “as described in ” . Please check.
7 Please review the symbols in the legend of Figure 1, to deter-
mine to us how they should be written, concerning the font
and the capitalization.
8“rad3” is written in 3 different ways till this page, please deter-
mine what each occurrence represents: a gene, a protein, ...,
to help us determining the font we have to use, please note
that, as you know, if a protein and gene have a symbol in
common, the gene is distinguished by italic font and all let-
ters are capitals.
9“ssl2” is written in 2 different ways. Please check.
10rtt has to be capitalized as it is an acronym, is it ok?
11Please check this symbol.
12Should we add “such” after “likelihood” .
13Please spell out AMB
14Please provide the issue number for reference .