Nucleotide Excision Repair: Variations

Associated with Cancer Development

and Speciation
J E CLEAVER1 .I R SPEAKMAN2. J P G VOLPE1
1 Laboratoryof Radiobiology and Environmental Health, University of California, San
Francisco, California 94143-0750; 2Depart17lent ofZnology, University ofAberdeen, Aberdeen
AB92TN

Introduction
Mechanism of nucleotide excision repair
The coupling of transcription with repair
Diseases of nucleotide excision repair
Xeroderma pigmentosum
Cockayne syndrome
Trichothiodystrophy
Interindividual variability of nucleotide excision
DNA excision repair and speciation
Implications for cancer and other health effects
Summary

I~TRODUCTIO~

The genome of cells presents massive problems of fidelity and maintenance of

accurate information (Lindahl, 1993). A diploid human cell has 1.3 X 10 10
bases that must be faithfully maintained, replicated and passed on to the
daughter cells. Given the large number of endogenous and exogenous
genotoxic agents that cells encounter during the lifetime of their host, it is not
surprising that such hosts have evolved elaborate means of safeguarding their
genomes. Although preventive mechanisms are by no means trivial (eg anti
oxidants), DNA repair is probably the most important mechanism a cell has to
maintain its genome faithfully. Major roles for repair systems have been recog
nized in mending endogenous oxidative damage in nuclear and mitochondrial
DNA, in correcting DNA mismatches associated with non-polyposis colon can
cer, in immunodeficiencies, in skin carcinogenesis in xeroderma pigmentosum
(XP) and in developmental and neurological abnormalities in XP, Cockayne
syndrome (CS) and trichothiodystrophy (TTD) (Cleaver and Kraemer, 1989).
Less obvious, but possibly equally important, are variations in repair between
individuals and between species.

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1995 Imperial Cancer Research Fund. 0-87969-469-6/95. $5.00 + .00 125
126 J E Cleaver. J RSpeakman. J PG Volpe

Repair systems range from those with high specificity, such as photolvases
and uracil-DNA glycosylase, to those with great versatility, such as nucleotide
excision repair (Sancar and Sancar, 1988). Photoreactivation directlv reverts
cyclobutane pyrimidine dimers formed by ultraviolet (DV) light to normal
nucleotides by phototransduction. This has a wide distribution among species,
but for unexplained reasons is missing in a variety of microorganisms such as
Bacillus subtilis and Schizosaccharomyces pombe and eutherian mammals in
cluding humans (Yasui et al, 1994). Dracil-DNA-glycosylase is widespread and
corrects uracil produced by deamination and misincorporation, by a base exci
sion mechanism (Sancar and Sancar, 1988; Cleaver and Layher, 1995). Mis
match repair removes single mismatched bases or small loops created by
replication slippage and is responsible for much tumour associated DNA se
quence variability (Modrich, 1994). Double strand breaks in DNA are rejoined
by non-homologous recombination in mammalian cells involving proteins that
bind specifically to DNA ends. Defects in some of these proteins result in
defective immunoglobulin rearrangement, and some circulating autoimmune
antigens are directed against these proteins (eg the Ku antigen) (Taccioli et al,
1994; Finnie et al, 1995).
Nucleotide excision repair (NER) removes photoproducts, bulky adducts
and many other damaged regions of DNA (Sancar, 1994). This seems to be the
most versatile way a cell has to repair its DNA, and this pathway is active when
many of the other pathways are overloaded with damage. A great deal of prog
ress has been made in recent years in the mechanism of nucleotide excision
repair, its coupling with transcription, the complex diseases exhibited by muta
tions in NER genes and variations between individuals and species (Hanawalt,
1994; Sancar, 1994). Consideration of this process not only highlights an ex
quisite molecular mechanism, but also illustrates a broad range of intriguing
questions in cancer and development.

Mechanism of Nucleotide Excision Repair

Early views on NER developed from the study of the capacity of cells to mend
damage produced in DNA by DV light, either DVC (240-290 nm) or DVB
(290-320 nm) (Cleaver and Mitchell, 1993). The most prevalent photo
products induced are the cyclobutane pyrimidine dimer and, at about 25% the
frequency, the pyrimidine-pyrimidone [6-4] photoproduct (Mitchell and
Nairn, 1989; Mitchell and Cleaver, 1990). The [6-4] photoproduct can further
undergo a DVB dependent conversion to its valence photoisomer, the Dewar
pyrimidone (Taylor and Cohrs, 1987). The distribution of these photoproducts
in human chromatin depends on base sequence, secondary DNA structure and
DNA-protein interactions. Pyrimidines flanking a dipyrimidine sequence in
crease photoproduct yield; purines suppress the yield. Photoproducts mapped
to nucleosome structure indicate dimers are formed preferentially on the out
er face of the DNA helix, but [6-4] photoproducts are much more common in
linker than core DNA (Gold and Smerdon, 1990; Mitchell et al, 1990). The
 Nucleotide Excision Repair 127

ratio of cytosine-containing cyclobutane dimers to thymine dimers increases

significantly within the UVB region :\fitchell and Cleaver, 1990). Dimers con
taining cytosine and [6-4J photoproducts, which are preferentially induced at
TC sequences, may have a major role in UVB (solar) mutagenesis, producing
C to T and CC to TT transition mutations (Brash and Haseltine, 1982; Wood
et al, 1984; Brash et al, 1987; Dumaz et al, 1993).
Damage by UV light has been mapped at nucleotide resolution in vivo
using the ligation mediated polymerase chain reaction and has shown that the
binding of transcription factors to promoter regions can both enhance and
suppress photoproduct formation at specific sites (Tomaletti et al, 1993, 1994;
Tornaletti and Pfeifer, 1994). The propensity of photoproducts to be formed in
transcription factor binding sites would serve to enhance mutations affecting
gene regulation and focus these mutations on specific tissues.
Photoproducts produced in DNA by UVC or UVB radiation are repaired
by a complex multistep process involving many interacting gene products (Fig.
1). These interactions can give rise to complex overlapping symptoms in
patients with mutations in these genes. The repair process involves removal of
a 27-29 nucleotide oligonucleotide that contains the photoproduct by
endonucleolytic cleavage 5 nucleotides on the 3' side of the photoproduct and
24 nucleotides on the 5' side (Sancar, 1994). The single strand region is pro
tected by Single strand binding protein (HSSB) and the gap is filled in by DNA
polymerase () or E, proliferating cell nuclear antigen (PCNA) and ligase. These
processes can be considered as involving sequential steps of photoproduct
recognition, assembly of the excision complex, displacement of the excised
fragment and polymerization of the replacement patch, but many puzzles
remain.
Photoproduct recognition is achieved by specific binding of the XPA gene
product, a 273 aminoacid (30 kDa) protein. This protein appears to be rate
limiting for repair in human cells but the precise mechanism of recognition is
unknown. Its greater binding coefficient for [6-4J photoproducts over dimers
influences the overall rates of repair such that [6-4J photoproducts are
removed much faster than dimers (Jones and Wood, 1993; Asahina et al,
1994).
The XPA protein bound to a photoproduct acts as a site for binding two
nucleases: the XPG protein that cuts 3' to the dimer and a heterodimer of
XPF and ERCC1 that cuts 5' to the dimer. Contact sites have been
demonstrated between XPA and ERCC1 and ERCC1 and XPF, but XPG may
act as an independent nuclease. Interestingly, another photoproduct binding
protein, XPE, appears to playa minor part in quantitative levels of repair, but
when it binds a photoproduct, it creates a DNase I hypersensitive site precise
ly where the XPG nuclease cleaves DNA (Reardon et al, 1993). Even so, the
role of the XPE gene product in repair remains a mystery: it is present in ex
cess over XPA, but is not required for repair in vitro. The damaged region is
unwound by the action of transcription factor TFIIH, which contains both 3'
5' (XPB) and 5' -3' (XPD) helicases. At least two components of TFIIH,
128 J E Cleaver. J RSpeakman. J PG Volpe

24nt 5nt
.. to . . . .

5'

~.I..J...LI..LJ....L.l..J..l...l...LJ....LL./"""".,,c...--~LL..LJ.....LL 5'
3'

TFIIH
B,D, ...
@HHR23B)
Fig. 1. Nucleotide excision repair: variation associated with cancer, development and specia
tion. HHR23B = RAD23B

XPB and XPD, interact with TP53 and initiate a signal cascade in damaged
cells that leads to cell cycle arrest and/or apoptosis. This functional process re
quires about 100 nucleotides of DNA along which to operate (Huang and San
car, 1994). This nuclease complex appears to have ready access to transcrip
tionally active regions of the cell, but access to non-transcribed regions, or the
non-transcribed strand, requires an additional set of gene products: the
XPC/RAD23B heterodimer (Masutani et al, 1994).

The Coupling of Transcription with Repair

The first evidence that excision repair might have a special relationship with
transcription was the discovery that cell lines from patients with Cockayne
syndrome had a genetically determined deficiency in the recovery of transcrip
tion after UV irradiation (Lehman et al, 1979). Although these cells also fail to
recover DNA replication, this second aspect of their phenotype has received
little attention. The next discovery was that the transcriptionally active genes in
normal cells were repaired faster than the rest of the genome and that the
transcribed strand was better repaired than the non-transcribed strand (Mel
lon et al, 1986). In expressed genes, a strand bias of mutations due to the per
sistence of damage on the non-transcribed strand has been observed (Dumaz
et al, 1993). An interesting question, therefore, is whether the lack of repair in
non-transcribed regions of the genome means that these regions accumulate
genetic variations that can emerge precipitously when cells enter altered states
of gene expression through development or through translocation of promoter
and enhancer regions.
The relationship between repair and transcription involves two main sets
of gene products. One set of gene products, those of the Cockayne syndrome
genes (CKN] and CKN2), are human analogues of the mutation frequency
decline locus in Escherichia coli and couple RNA polymerase II transcription
to the excision complex (Venema et al, 1990a; Hanawalt, 1994). The precise
 l'Jucleotide Excision Repair 129

TABLE 1. Nucleotide excision repair: genes identified in human disorders and UV

sensitive Chinese hamster ovary ((HO) cells

Human Rodent Chromosome Relative

group groupa location repair (%) Function
Xeroderma pigmentosum
A 9q34.1 <2 photoproduct binding protein
Bb ERCC3 2q21 3-7 3' to 5' helicase (TFIIH)
C 3p25.1 10-20 transcription independent repair
Db,c ERCC2 19q13.2 25-50 5' to 3' helicase (TFIIH)
E 11 40-50 photoproduct binding protein
Fd ERCC4 16p13 10-20 nuclease"
G ERCC5 13q32-33 <2.25 nuclease"
Variant 100 replication factor

Cockayne syndrome
A 100 transcription coupling factor
B ERCC6 lOq21.1 100 transcription coupling factor

Trichothiodystrophy
A 15 TFIIH component

Unidentified syndrome
ERCCl 19q13.2 0 nuclease"

"Cenes in the ERCC series are found in human and rodent cells, and were first identified through
selection of UV sensitive hamster cells. ERCC1 does not correspond to any Xl' group. There are eight or
m9,re CHO complementation groups, but the higher numbered groups are rare and still being characterized
Patients also exhibit symptoms commonly associated with Cockayne syndrome: dwarfism, cutaneous
features and mental retardation
cSome patients have symptoms of trichothiodystrophy
dThis chromosome assignment is made for the ERCC4 gene, its identity to XPF is not yet firm
eThe ERCCl and ERCC4 genes are subunits of one of the excision nucleuses, XpG/ERCC5 is another
excision nuclease

molecular definition of this "coupling" has yet to be explained. Cells carrying

mutations in these genes show normal levels of overall genome repair and only
lack the enhanced repair of the transcribed strand. One hypothesis is that
these genes, one of which (C KN2) is a helicase, act to move RNA polymerase
II back from transcription stop sites (Hanawalt, 1994).
The other involvement with transcription concerns the basal transcription
factor, TFIIH (Schaeffer et al, 1993). This factor regulates basal transcription
by RNA polymerase II, and most of its eight peptide components have second
functions in DNA repair. Two of the helicases in TFIIH correspond to the
XPB (Weeda et al, 1990) and XPD (Weber et al, 1990) genes of xeroderma pig
mentosum, one involves TTDA (Bootsma and Hoeijmakers, 1994) and others
are known to playa part in repair from their analogues in the yeast transcrip
tion factor b. The extent to which the individual components of TFIIH or fac
tor b act as a tight complex, a loose dynamic association or separate into indi
vidual components is unknown and may differ between humans and yeast. An
attractive idea is that the stalling of polymerase II at a damaged site may be a
130 J E Cleaver. J RSpeakman. .I PG Volpe

mechanism to deliver repair helicases such as XPB and D to the locality of a

damaged site for unwinding of the helix, but TFIIH is not known to be associ
ated with RNA polymerase II during the transcription elongation. This idea
cannot, however, account for the fact that most patients with mutations in
TFIIH genes have low repair of both transcribed and non-transcribed regions
of the genome; TFIIH or its components must therefore have an additional
role in repair of the overall genome independent of the transcriptional activity
of the DNA.
Non-transcribed regions of the genome also require the activity of a
specific heterodimeric protein coded by the XPC and RAD23B (formerly
HHR23B) genes, although its precise function is unclear (Masutani et al,
1994). Deficiencies in XPC result in the total repair of a cell being reduced to
10-25% of normal. This repair is non-uniform, being clustered in transcribed
genes and consisting of tandem groups of repair patches (Karentz and Cleaver,
1986; Venema et al, 1990b). Since actively transcribed genes involve much less
than 10% of the total genomic DNA, the repaired regions must involve longer
stretches of DNA around actively transcribed genes.

Diseases of Nucleotide Excision Repair

Many of the components of the excision repair process are the products of
genes that give rise to a variety of sun sensitive and developmental disorders
when they carry mutations (Table 1). The complexity of these diseases comes
not only from the specific role that the gene products have in repair, but also
from second roles in transcription factors and signalling cascades. In addition
to the TFIIH components and the CSA, B coupling factors, some gene pro
ducts have other interactions. XPB interacts with TP53 (Greenblatt et al, 1994)
and PCNA with GADD45 (Smith et al, 1994), both of which link the repair
process with extensive networks of intracellular signalling and cell cycle con
trol.
Genes involved in nucleotide excision repair have been associated with a
variety of human disorders that appear to regulate the response of mammalian
skin to sunlight damage and playa part in neurological and developmental dis
orders (Kraemer et ol, 1987). These include the genes associated with XP (ap
proximately eight complementation groups), Cockayne syndrome (approxi
mately two complementation groups) and trichothiodystrophy (approximately
three distinct phenotypic types) (Cleaver and Kraemer, 1989). It is known
from selection of UV sensitive cell lines and from analogues in yeast that
several other genes playa part in DNA repair, but these have not been seen in
human disorders. These include ERCC1, RAD23B and RAD6A,B. ERCCl
knockout mice have been raised and at first exhibited a fatal neonatal liver dis
order (McWhir et al, 1993), but on other genetic backgrounds, animals with
this mutation live for up to 6 months. The complex disorder categorized as
TID may have additional clinical manifestations as genetic disorders of devel
opment involving one or more of the clinical symptoms but without photosen
 Nucleotide Excision Repair 131

sitivity or repair deficiency Bootsr..s and Hoeijmakers, 1994). The XPD gene
is particularly complex, with pa;::c-n:s exhibiting either XP, XP with neuronal
loss, XP with CS, XP with TID. or CS or TTD alone. To set the scene for con
sidering these complexities, a brier' description of the individual, separate dis
orders ofXP, CS and TTD is helpful. Complex and mixed symptoms may then
be viewed as the result of mutations that affect protein-protein interactions
(Friedberg, 1993).

XERODERMA PIGMENTOSUM
Xeroderma pigmentosum is a rare autosomal recessive disease that occurs at a
frequency of about 1:250 000 in the USA (Kraemer et al, 1984, 1987; Cleaver
and Kraemer, 1989). Affected patients (homozygotes) have sun sensitivity
resulting in progressive degenerative changes of portions of the skin and eyes
exposed to the sun, often leading to neoplasia. Some XP patients have, in addi
tion, progressive neurological degeneration. Obligate heterozygotes (parents)
are generally asymptomatic. The median age of onset is 1-2 years of age, with
skin rapidly taking on the appearance of that seen in individuals with many
years of sun exposure. Pigmentation is patchy and skin shows atrophy and
telangiectasia with development of basal and squamous cell carcinomas. The
frequency of cancers is 2000 times that seen in the general population under
20 years of age, with an approximate 30 year reduction in lifespan. Squamous
cell carcinomas and melanomas occur with elevated rates and occur with a
body distribution similar to that in normal individuals. Internal cancers seem
only slightly elevated if at all, consistent with the view that the disease is not
due to a failure to repair spontaneous damage but only exogenous carcinogen
induced damage.
There are eight genes identified among XP patients: seven excision repair
defective (XPA-G) and one replication defective. Cells from patients with XPA
to G all fail to excise pyrimidine dimers, [6-4] photoproducts and other car
cinogen adducts from DNA. By contrast, the variant appears to have normal
excision repair. These XP genes are all involved in the primary damage recog
nition and excision complex (Fig. 1). Particularly intriguing is the relationship
of repair deficiency to the neurological deficiency seen in a Significant fraction
of XP patients, especially those with mutations in XPA, B, D and G genes.
There may be two kinds of neurological problems in XP: the Simple neurologi
cal decline seen in XPA and some XPD patients and the more complex
neurological and developmental disorders (XP-CS syndrome) seen in some
XPB, D and G patients.
Patients with mutations in XPA lack a functional copy of the primary
photoproduct specific binding protein (Satokata et al, 1992a,b,c; Asahina et al,
1994) and are extremely sensitive to sunlight, but they also show progressive
neuronal loss. They may be born microcephalic and mentally retarded, or lose
mental faculties in early life. The XPA neurological decline may be due to a
132 J ECleaver. J RSpeakman. J PG Volpe

failure to repair DNA damage in the brain that is caused by high rates of
oxidative metabolism (Satoh et al, 1993). Such damage might be manifested as
neuronal loss in the non-dividing neurons, which are among the most trans
criptionally active cells of the body. This neurological disorder may be dif
ferent from that seen in patients whose genes code for components of TFIIH
where neurological and developmental disorders may be more complex and
correspond to a family of "transcription factor disorders".
These more complex disorders are illustrated by the patients. One of the
relatively common complementation groups, XPD, is one of the most complex
(Weber et al, 1990). Patients with mutations in the XPD gene have been iden
tified with symptoms of XP alone, XP plus neurological decline similar to XPA,
XP plus the developmental aspects of CS or TID and most recently of TTD
without photosensitivity. The most likely explanation for this complexity lies in
the protein's place in the multiprotein complex of TFIIH such that mutations
can affect either the protein alone or the protein contacts with other factors in
volved in disparate functions. Mapping mutations with tertiary structures in
macromolecular complexes will be a long difficult project, but should help
solve this problem of phenotypes. Mutations reported in this XPD gene are
complex involving multiple genetic changes in the alleles, perhaps represent
ing the accumulation of several genetic events before the disease is
manifested.
One of the largest groups, XPC, is often referred to as the common or clas
sic form and patients rarely show symptoms other than cancers. One charac
teristic of repair unique to this group is that the reduced repair consists of lo
cal regions of normal repair in a large background of unrepaired genomic
DNA. This residual repair occurs in regions around transcriptionally active
genes and the repair deficiencies involve non-transcribed regions and the non
transcribed strands of active genes (Karentz and Cleaver, 1986; Venema et al,
1990b). This raises the dilemma that high rates of cell killing, somatic mutation
and cancer from UV light in XPC patients are associated with repair deficien
cies in the non-transcribed regions of the genome. Mutations in expressed
genes arise from unrepaired lesions in the non-transcribed strand (Dumaz et
al, 1993).
The mechanism underlying the XP variant cells is another puzzle. Patients
are very similar to XPC patients and are not clinically distinctive (Cleaver and
Kraemer, 1989). But the defect in this group does not seem to be associated
with an obvious excision repair defect. These cells appear near normal in their
ability to excise DNA damage and only show a slight sensitivity to UV light.
The cells instead show a reduced ability to replicate damaged DNA. One pos
sibility is that the XP variant could involve mutations in a replication factor
responsible for fidelity in replication. An attractive candidate is HSSB because
it is involved in both repair and replication and mutants in the corresponding
gene in E coli are UV sensitive. Transformation ofXP variant cells by SV40 en
hances several phenotypes including UV sensitivity in the presence of caffeine
and sister chromatid exchanges (Volpe and Cleaver, in press), again implicat
 Nucleotide Excision Repair 133

ing replication factors. It is PUZW=-lS That the genetic defect responsible for the
slight reduction in UV resistance ir. V variant cells is stilI capable of produc
ing high mutation rates and the se\-ere clinical symptoms shown by XP variant
patients.

COCKAYNE SYNDROME
Cockayne syndrome is an autosomal recessive disease characterized by cachec
tic dwarfism, retinal abnormalities, microcephaly, deafness, neural defects and
retardation of growth and development after birth. Carcinomas of the skin as a
result of hyperphotosensitivity are not seen in patients with CS, setting this
disease apart from XP. There are two genes associated with CS alone, CKNl
(formerly CSA) and CKN2 (formerly CSB), of which CKN2 is a helicase and
CKNl belongs to the WD repeat class (Neer et al, 1994). The excision of DNA
photoproducts from total genomic DNA of CS cells is normal, but repair of
the transcribed strand in transcriptionally active genes is reduced to the level
of the overall genome.
A significant number of patients do not fit the standard definition of CS or
XP. Two XPB families have been identified, for example, that have both CS
symptoms and XP excision defects and the gene product is part of TFIIH
(Weeda et al, 1990; Schaeffer et al, 1993). A few XPD and XPG patients also
have CS symptoms. This association can be explained for XPD, which is within
TFIIH, but it requires hypothesizing that the XPG nuclease also interacts with
the transcription factor. A series of patients has recently been summarized in
which the clinical presentation includes photosensitivity, but either they have
no easily classified syndrome or their photosensitivity is atypical for the
syndrome they do exhibit (Cleaver and Thomas, 1993). Their cellular
properties have had little biochemical investigation, but a feature common to
most is that the cells are UV sensitive and that their recovery of RNA tran
scription after UV irradiation is reduced, even though measurements of DNA
repair over short times after irradiation are within the normal range. The
biochemical features of these cases therefore overlap with some of the features
of CS, although they do not have the well defined characteristics of this partic
ular disorder. The manifestations of mutations in these transcription coupling
genes are therefore much more varied and many more gene products than
CKN 1 and CKN2 may be involved in the recovery of transcription from UV
damage.

TRICHOTHIODYSTROPHY
Trichothiodystrophy is a rare autosomal recessive disorder characterized by
sulphur deficient brittle hair and ichthyosis (Lehmann et al, 1988; Broughton
et al, 1990). Hair shafts split longitudinally into small fibres, and this brittle
ness is associated with levels of cysteine/cystine in hair proteins that are 15
50% of those in normal individuals. The condition is also accompanied by
physical and mental retardation of varying severity. The patients often have an
134 J E Cleaver. J RSpeakman. J PG Volpe

unusual facial appearance, with protruding ears and a receding chin. Mental
abilities range from low normal to severe retardation. The full spectrum of
symptoms is described under the initials PIElDS-photosensitivity, ichthyosis,
brittle hair, immunodeficient syndrome. Patients are known with a subset of
these symptoms and ones lacking the photosensitivity might correspond to
mutations affecting proteins or sites that are not involved in DNA repair. The
most severe TTD cases with photosensitivity, repair deficiencies and
neurological complications correspond to mutations in the XPD gene. Others
show reduced repair without photosensitivity or normal UV responses. Some
of these correspond to a TTDA gene, which is a component of TFIIH, whereas
others are distinctive mutations in the XPD gene despite normal repair
(Bootsma and Hoeijmakers, 1994).

I~TERI~DIVIDUAL VARIABILITY OF ~UCLEOTIDE EXCISIO~

Repair systems vary considerably between cells, tissues, individuals and

species. Some processes such as photoreactivation may be completely missing
from human cells, others such as 0 6 alkyltransferase are much more active in
liver than in brain and others such as nucleotide excision repair can be much
more active in human cells than in rodent cells. Variations between individuals
in radiation sensitivity and in repair have been observed, but one of the more
systematic studies was carried out using a reporter gene, CAT (chlorampheni
col acetyltransferase}, and correlations between age, cancer risk and other vari
ables have been noted (Athas et al, 1991; Wei et al, 1993, 1994a,b).
The reporter gene was irradiated as a plasmid vector prior to transfection
into lymphocytes from a variety of donors. Since residual CAT function was as
sayed, this essentially measured the host cell's capacity for repair of an actively
transcribed extrachromosomal gene. Results are not necessarily quantitatively
identical to repair of a transcribed endogenous chromosomal gene or the bulk
of the non-transcribed regions of the genome. In large surveys (Wei et al,
1993, 1994a,b), the residual CAT activity (usually in the range of 5-10% of
unirradiated CAT vector) after a standard UV dose (700 J/m 2 ) was -determined,
which raises a potential technical problem. Since CAT inactivation by UV
damage is exponential, with significant breaks in the curve to a more resistant
tail at high doses, the residual CAT activity is neither invariant with respect to
dose nor directly proportional to repair capacity. In fact, if repair capacity
determines the slope of the CAT inactivation curve, as comparisons of XP and
normal cells indicate, then changes in the residual activity at a fixed dose will
overestimate variations in repair. For example, a doubling of residual CAT ac
tivity from 5% to 10% can be caused by an increase of only a third in the D 37
(37% survival dose). In most inactivation curves, a measure such as D 37 would
have been a better and a dose independent measure of repair capacity, espe
cially for exponential or one-hit curves.
Given these caveats, this technique has been applied to several human
 Frr

Nucleotide Excision Repair 135

populations (Wei et al, 1993; 199-a.b. Residual CAT activity declines with
age, is generally lower and not age dependent in patients with basal cell cancer
and is also lower in patients with pour tanning or a burning response to sun ex
posure. Women with low residual C\ T activity were much more at risk for
basal cell cancer than men. Although these results may, for the reasons dis
cussed, overestimate quantitative aspects of the variations in repair, they do
spotlight nucleotide excision repair capacities in active genes as factors in hu
man cancer.

DNA EXCISION REPAIR AND SPECIATION

There are also undoubtedly real differences in the capacity of cells from vari
ous mammalian species to carry out NER of UV damage. Repair in rodent
cells, for example, is much less than that in human cells. Unscheduled
synthesis and photoproduct excision after UV exposure, for example, are about
tenfold less in mouse, rat and hamster cells than in human cells (Hart and Set
low, 1974; Kato et al, 1980; Francis et al, 1981). Since unscheduled DNA
synthesis is usually measured by a 2-4 hour labelling period immediately after
irradiation, this assay measures the initial rates of more rapidly excised
photoproducts. The reported differences between species therefore mainly
represent the excision of [6-4] photoproducts from the whole genome and
cyclobutane dimers from active genes (Mitchell et al, 1985; Mitchell, 1988;
Cleaver, 1989). Cyclobutane dimers are excised much more slowly from non
transcribed regions of the genome and consequently contribute less to overall
unscheduled synthesis. The rates of excision of dirners from actively tran
scribed genes are closer between different species, but human repair still ex
ceeds that of rodents (Bohr, 1991; Terleth et al, 1991).
What remains less clear, however, is the molecular basis for these dif
ferences and what their consequences are for the whole animal. The molecular
basis for these differences is becoming clear now that the functions of various
gene products in repair have been identified. The XPA photoproduct binding
protein, for example, is rate limiting for repair in the whole genome, but has a
preference for directing repair first to transcriptionally active regions, due
presumably to competition with chromatin proteins bound more tightly in
non-transcribed regions of the genome (Cleaver et al, in press). The
XPC/RAD23B dimeric protein, however, seems to be rate limiting for repair
of non-transcribed regions of the genome (Masutani et al, 1994). It is possible
that this factor restricts the amount of repair in non-transcribed regions of the
genome in mouse cells more than in human cells.
The significance of variation in the repair of the overall genome for dif
ferent species is less clear. The first study of a few species by Hart and Setlow
(1974) claimed a correlation between the lifespans of species and overall DNA
repair and this topic has been controversial ever since (Kato et al, 1980; Fran
cis et al, 1981; Bernstein and Bernstein, 1991). The most extensive survey was
136 J E Cleaver. J RSpeakman. J PG Volpe

that of Kato et al (1980) who analysed UV induced repair in 34 mammalian

species of 11 orders using autoradiographic techniques. These data showed
wide variations in repair with no obvious biological correlates. The data set of
Kato et al (1980) remains, however, the most extensive and taken at face value
is well worth a re-examination by more thorough statistical techniques.
The most suitable approach for such data is the method of residuals. We
have analysed (Speakman J, unpublished) possible correlations of repair to
diploid DNA content, lifespan, diet, nocturnality and phylogenetic relatedness,
all of which can be obtained or inferred from the original data. Using this
statistical technique, the results revealed that NER is related to genome size
and phylogenetic relationships (closely related species had similar rates of
repair), but bears no relationship to diet, activity and lifespan. These two posi
tive relationships shed interesting light on the organization and evolution of
repair. Repair increased roughly in relation to the square of genome size. This
relationship can best be understood as due to two effects that increase with
genome size: there is more DNA to be damaged and a larger volume of DNA
to search (Lambert et al, 1992). After correction for DNA content, further
analysis showed that animals that repaired their genomes well, relative to the
genome size, did not live longer than might be expected from their body size.
We did, however, find that species of mammals have DNA repair rates very
similar to those of other species in the same genus and to some extent also the
same family. The similarity between any two species declines as they become
progressively less related. The absence of other factors in this data set raises
the question of whether the variations in cancer risk with repair among human
donors (Wei et al, 1993, 1994a,b) occur strictly within species, and other fac
tors dominate risks for comparisons between species. Particularly interesting
will be results from gene transfer experiments in which rodent repair is raised
to human levels and the biological consequences determined. This might pro
vide information about whether there is any value to the repair of the majority
of the genome, the untranscribed regions.

IMPLICATIONS FOR CANCER AND OTHER HEALTH EFFECTS

When first described, XP presented a simple relationship for reduced DNA
repair leading to increased mutation rates and accumulated mutations
eventually leading to cancers (Cleaver and Kraemer, 1989). As a more detailed
understanding of XP and related repair deficient disorders has been reached
and of other genetic predispositions to cancer involving oncogenes and tumour
suppressor genes, Simple relationships no longer hold. Problems remain in un
derstanding both heterozygote and homozygote symptoms associated with
repair deficiency.
Patients with XP have more than a lOOO-fold increased risk of developing
cancer, getting cancer much earlier than the general population. In this
regard, they are similar to patients with other inherited cancer syndromes, in
 Nucleotide Excision Repair 137

cluding patients carrying a mutated tumour suppressor gene such as TP53 or

RB or the mismatch repair gene MSH2 (Friend et al, 1987; Sager, 1989; Par
sons et al, 1993). Since some XP patients show little if any ability to repair their
DNA, these statistics are not surprising. However, when XP homozygotes are
protected from sunlight and cigarette smoke, it is surprising that they show
little if any Significant increase in cancer rates (Kraemer et al, 1984) despite
the fact that every one of their cells is missing two alleles essential for
maintaining their genomes. By contrast, retinoblastoma and hereditary non
polyposis colon carcinoma (HNPCC) patients are heterozygotes, lacking only
one single allele. Cancer in these patients is associated with loss of the second,
functionally normal gene. Further mutations are then necessary before a cell
can become neoplastic, as only the rare cell becomes neoplastic in TP53-/
homozygous mice (Donehower et al, 1992; Lee et al, 1994). If the cancer
syndrome patients who inherit only a single defective allele go on to develop
cancer, why are protected XP patients who inherit two defective alleles so un
likely to develop cancer spontaneously? Several explanations are likely. One is
that the excision repair deficiency of XP has a minor role in the repair of low
levels of spontaneous, endogenous damage to internal tissues, much of which
can be repaired by base excision mechanisms. The brain may be a unique tis
sue where high oxidative metabolism and the possibility of DNA damage occur
in non-dividing neurons, with the result that neuronal loss rather than cancer
occurs. Secondly, sun exposure corresponds to a very high exposure to a DNA
damaging agent. The 37% survival dose of unprotected normal human cells in
culture is of the order of 30 minutes of direct sunlight. Many cells in the skin
of an XP patient consequently receive lethal doses resulting in a stimulus for
proliferative hyperplasia. This dose rate may be a factor in the tumour promot
ing action of sunlight which supplements its mutagenic action. Such conditions
do not apply to cells of internal tissues.
The repair deficiencies seen in XP, CS and TTD in fact bear a surprising
relationship to the skin cancer and the other characteristic central nervous sys
tem and developmental disorders. Skin cancer is seen in those patients in
which repair of the non-transcribed regions of the genome is defective,
whether or not transcribed regions are also affected. Other central nervous
system and developmental symptoms are seen when repair of transcribed
regions of the genome is defective, whether or not the rest of the genome is af
fected. This latter observation has led to a postulated class of "transcription
syndromes" (Bootsma and Hoeijmakers, 1994). The relevance of this observa
tion is that repair deficiencies in XP may result in the accumulation of damage
and mutations in unexpressed regions of the genome, which emerge
precipitously after reaching a critical level or when other genomic changes oc
cur.
Many tumour suppressor genes, unlike the XP genes, contribute to cell
cycle control or regulation of genomic stability in response to spontaneous
replication errors. Mutations in TP53 are found in the vast majority of human
squamous cell carcinomas. Ziegler et al (1994) have hypothesized that
138 J E Cleaver. J RSpeakman. J PG Volpe

TP53+/- cells have a selective advantage over TP53+/+ (wild type) cells in the
presence of UV. This is because UV damaged wild type cells readily undergo
apoptosis, but TP53+/- cells are somewhat defective and TP53-/- cells appear
even more defective in this pathway. This allows for TP53 deficient cells to ex
pand in the population of skin cells so that the probability that additional
mutations occur within this lineage is greatly increased. Such increases will be
even greater in an XP homozygous individual. Such reasoning suggests another
question which is why XP heterozygotes have little if any elevated skin cancer
rates (Swift and Chase, 1979). These individuals do not avoid the sun and yet
all of their cells are XP+/-. Surely, some of their skin cells mutate to XP-/
from UV induced damage or spontaneous mutation. Such sun damaged repair
defective cells should have a greatly elevated risk of acquiring additional
neoplastic mutations, including TP53. Why then do such mutated cells in XP
heterozygous individuals seldom appear to go on to form tumours? The reason
is probably simply numerical. XP homozygous cells will appear rarely in the
skin of XP heterozygous individuals and the probability of an initiated
neoplastic cell going on to develop into a tumour is also very rare (Thomassen
et al, 1985; Miller et al, 1987). Even with a mutation rate from UV exposure
that is several orders of magnitude higher than normal in XP homozygous
cells, most of the homozygous cells appearing in the skin of a heterozygote will
be preferentially killed by UV rather than lead to cancer. This would be con
sistent with the observed absence of elevated cancer risks in heterozygous in
dividuals. The carrier state for NER pathways specific for exogenous sources
of damage is less likely to be a major contributing factor to human cancer than
those dealing with spontaneous and endogenous sources of genetic stability.

SUMMARY
Nucleotide excision repair requires the action of multiple interacting proteins
that locate damage in DNA, remove it as a short oligonucleotide and
synthesize a replacement patch. Mutations in genes coding for these proteins
give rise to a wide range of diseases involving skin carcinogenesis, neuronal
decline and developmental disorders of bone and central nervous system.
Complex clinical symptoms of more than one clinical disorder may occur be
cause of mutations that influence protein-protein interactions. Significant dif
ferences in repair occur between individuals and species for which the
molecular basis and phenotypic consequences have yet to be explained.

Acknowledgements
This work was supported by the US Department of Energy, Contract DE
AC03-76-SFOl0l2 and by the National Institutes of Health National Research
Service Award 5 T32 ES07106 from the National Institute of Environmental
Health Sciences (JPGV).
 Nucleotide Excision Repair 139

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