Environmental and Molecular Mutagenesis 37:329 –339 (2001)
XPA Protein Alters the Specificity of Ultraviolet
Light–Induced Mutagenesis in Vitro
Nicole M. King, Gregory G. Oakley, Mario Medvedovic,
and Kathleen Dixon*
Department of Environmental Health, University of Cincinnati College of Medicine,
Cincinnati, Ohio
Studies of ultraviolet (UV) light mutagenesis have
demonstrated mutations at common sites in the
target genes of shuttle vector plasmids replicated in
cultured cells or by cellular extracts. The reasons
for the specific pattern of mutagenesis are largely
unknown. We have examined the specificity of
UV-induced mutagenesis by replicating plasmid
pLS189, irradiated with 40 J/m2 UVC or unirradi-
ated, in either xeroderma pigmentosum group A
(XP-A) or HeLa cellular extracts. The XP-A extract
displayed slightly lower replication ability, but pro-
duced a higher mutant frequency, compared to
that of HeLa extract. Use of irradiated plasmid
inhibited replication by an average of 63% and
increased the mutant frequency by an average of
16.7-fold. Analysis of mutation spectra revealed
nonrandom patterns of mutagenesis that differed
significantly between HeLa and XP-A extracts. In
comparison to HeLa extract, replication in XP-A
Key words: mutation spectrum; translesion replication; ultraviolet; xeroderma pigmentosum
INTRODUCTION
Xeroderma pigmentosum (XP) is a rare autosomal reces-
sive disease exemplifying the relationship between DNA
damage, repair, mutagenesis, and cancer. XP is character-
ized by extreme sun sensitivity, various degrees of neuro-
logical impairment, and more than a 1000-fold increase in
the frequency of skin cancer compared to that in normal
individuals [Kraemer et al., 1984]. Approximately 20% of
patients are XP variants (XP-V), which have been shown to
be defective in translesion replication attributed to a muta-
tion in the gene for polymerase ␩ [Masutani et al., 1999].
The remaining 80% of patients have a defect in nucleotide
excision repair (NER) and have been classified into seven
complementation groups (XP-A through XP-G) [Reardon et
al., 1993; Wood et al., 1993]. XPA codes for a 273 amino
acid zinc metalloprotein necessary for the damage recogni-
tion step of NER [Tanaka et al., 1990; Robins et al., 1991].
The XPA protein preferentially binds to UV-damaged over
undamaged DNA [Robins et al., 1991; Jones and Wood,
1993]. Binding of XPA to the RPA protein increases its
affinity for damaged DNA [Li et al., 1995]. XPA has also
been shown to form a complex with ERCC1 and ERCC4/
XPF [Park and Sancar, 1994]. TFIIH is recruited to the
repair complex by XPA [Park et al., 1995].
© 2001 Wiley-Liss, Inc.
extract resulted in lower frequencies of GC 3 AT
transitions and tandem double-base substitutions,
and a higher frequency of deletions. Replication in
HeLa extract produced hotspots at positions 100,
108, and 156 that were not produced by XP-A
extract. Furthermore, XP-A extract produced hot-
spots at positions 124, 133, and 164, sites not
characteristic of previous UV-induced mutagenesis
studies using XPA-expressing cells. Addition of pu-
rified XPA protein to reactions containing XP-A ex-
tract altered each of these parameters, including
loss of the hotspots at positions 124 and 133, to
yield a more HeLa-like spectrum. These results in-
dicate a previously uncharacterized role of the XPA
protein in influencing the specificity of UV-induced
mutagenesis during DNA replication. Environ.
Mol. Mutagen. 37:329 –339, 2001. © 2001
Wiley-Liss, Inc.
Cell lines derived from XP-A patients have been used in
a number of studies as repair-deficient backgrounds in
which to investigate the mechanism of UV-induced mu-
tagenesis [Bredberg et al., 1986; Parris et al., 1994; Levy et
al., 1996a,b]. These studies demonstrated that XP-A cells
replicate UV-damaged plasmids and fix mutations in the
supF gene in a pattern characteristic of UV. Similar patterns
of mutagenesis have also been observed following replica-
tion of UV-irradiated plasmid in CV-1 cells [Hauser et al.,
1986], in GM00637 cells [Bredberg et al., 1986], and in
CV-1 or HeLa extract in vitro [Carty et al., 1993, 1995]. The
present study was undertaken to test the ability of XP-A
extract to complete in vitro replication of an UV-irradiated
plasmid containing the supF gene. The frequency, location,
Grant sponsor: National Institutes of Health; Grant numbers: ES05400,
ES06096, and ES07250.
N. King is currently at University of North Carolina, Department of
Radiation Oncology, Chapel Hill, NC 27599.
*Correspondence to: Kathleen Dixon, University of Cincinnati, Depart-
ment of Environmental Health, 3223 Eden Avenue, Cincinnati, OH 45267-
0056. E-mail: kathleen.dixon@uc.edu
Original submission 5 February 2000; resubmitted 12 December 2000; and
accepted 19 February 2001
330 King et al.
and type of mutations fixed in replicated molecules are
presented. Although the pattern of mutagenesis in HeLa
extract is similar to that of previous reports, the results of
replication in XP-A extract reveal a significantly different
pattern of mutations. Furthermore, the specific hotspots of
mutagenesis identified in XP-A extract differ from those
identified in HeLa extract in this and a previous study [Carty
et al., 1995]. Addition of purified XPA protein to replication
reactions containing XP-A extract results in partial comple-
mentation of the novel spectrum. These results suggest that
the XPA protein functions, outside of its role in repair, as a
modifier of translesion synthesis to affect the specificity of
UV-induced mutagenesis.
MATERIALS AND METHODS
Cell Culture
The SV40-transformed fibroblast line GM04429, derived from a patient
with xeroderma pigmentosum (XP12BE, group A), was kindly provided by
Michael Seidman (National Institute on Aging [NIA], National Institutes of
Health [NIH]) and purchased from the NIGMS Coriell Cell Repository
(Camden, NJ). Coriell was also the source of the SV40-transformed fibro-
blast cell line GM00637G. LM217 cells and SV40-transformed fibroblasts
were a generous gift from Leon Kapp (University of California, San
Francisco). HeLa cells were grown in DMEM plus 10% FBS and 1%
penicillin/streptomycin (10,000 U/ml:10,000 ␮g/ml). XP12BE cells were
grown in the same medium, but with the addition of 1% L-glutamine (200
mM). For the colony-forming assay, LM217 and GM00637G cells were
grown in MEM plus 15% FBS, 2% amino acids, 1% nonessential amino
acids, 1% vitamins, and 1% penicillin/streptomycin (medium 1) or in
XP12BE-type medium as listed earlier (medium 2), as noted in Figure 1.
All cell lines were grown at 37°C in 5% CO2. All medium components
were purchased from Gibco/Life Technologies (Rockville, MD).
Plasmids
Plasmid constructs pLS189(96C) and pLS189(96G) [Levy et al., 1996a]
were provided by Michael Seidman (NIA, NIH). The sequence of pLS189
includes a pBR327 origin for replication in bacteria, the SV40 origin for
replication in mammalian cells or cell extract, an ampicillin-resistance gene
for selection of transformed bacteria, and the supF mutagenesis target
gene. These vectors are derivatives of pZ189 in which sequences surround-
ing positions 155 and 156 have been duplicated around positions 99 and
100. The plasmid was grown in the MBM7070 strain of E. coli [Seidman
et al., 1985] and purified using a Qiagen Maxiprep kit (Qiagen, Valencia,
CA). The plasmid was irradiated at 100 ng/␮l in TE buffer (10 mM
Tris–HCl/1 mM EDTA) using a low-pressure mercury lamp emitting
maximally at 254 nm at a dose of 40 J/m2, then diluted to 25 ng/␮l in TE
buffer for replication reactions.
Replication Extracts
XP12BE-SV cells (1.2 ⫻ 106) or HeLa cells (7.6 ⫻ 106) were plated per
150-mm dish and grown for 2 days. Cell extracts containing cytoplasmic
and soluble nuclear proteins were made from less than confluent cultures
by homogenization and hypotonic extraction [Li et al., 1984]. Data were
gathered from three separate XP12BE-SV extracts and two separate HeLa
extracts, such that results are not specific to a certain extract preparation.
Fig. 1. Post-UVC colony-forming ability. Cell lines and growth media
are indicated by the following symbols: LM217 in medium 1 (䡺) or
medium 2 (f), GM00637G in medium 1 (‚) or medium 2 (Œ),
XP12BE-SV lab stock in medium 2 (F—), and XP12BE-SV newly re-
ceived from the repository (f—). Points are the average of two to six
separate dishes, combined from two experiment dates. Bars represent
standard error.
XPA Protein
The purified XPA protein was a generous gift from Aziz Sancar (Uni-
versity of North Carolina, Chapel Hill). The human XPA cDNA [Park et
al., 1993] tagged with 6⫻ histidine in the pRSET vector (Invitrogen, San
Diego, CA) was overexpressed in E. coli DR153 and purified by the
procedure of Jones and Wood [1993]. The amount of XPA added to
replication reactions (25 ng) was chosen to be equal to or less than the
estimated amount of wildtype XPA protein in cellular extracts (60 ␮g total
protein) based on Western blot band intensity, the amount required to
complement repair activity of extracts [Park et al., 1995; Li et al., 1995],
and expression levels of XPA mRNA [Layher and Cleaver, 1997].
In Vitro Replication
DNA replication reactions were carried out in a volume of 30 ␮l
containing 60 ␮g of extract protein; 25 ng of plasmid; 30 mM Hepes (pH
7.5); 7 mM MgCl2; 0.5 mM DTT; 100 ␮M each of dATP, dGTP, dTTP; 50
␮M dCTP; 10 ␮Ci [␣-32P]dCTP; 200 ␮M each of GTP, UTP, CTP; 4 mM
ATP; 40 mM creatine phosphate; 10 ␮g creatine phosphokinase (bovine
heart); and 1 ␮g SV40 large T-antigen (Molecular Biology Resources,
Madison, WI). Some reactions also contained 25 ng purified His-XPA
protein. At intervals during a 3-hr incubation at 37°C, a 2.2-␮l aliquot of
each reaction was stopped by 30-min incubation with 2.2 ␮l 2⫻ Proteinase
K/EDTA (PK/EDTA) to a final concentration of 100 ␮g/ml and 15 mM,
respectively. The aliquots were spotted to each of two DE81 Whatman
filters (Whatman, Clifton, NJ), at 2 ␮l per filter. The filters were washed six
times in 0.5 M Na2HPO4/NaH2PO4 (pH 7.0) buffer. Incorporation was
measured by liquid scintillation counting. Replication was expressed as
picomoles of total incorporation per 30-␮l reaction based on a calculation
that includes the specific activity of the 32P on the experiment date. After
3 hr, replication reactions were stopped by incubation with PK/EDTA, as
earlier, and incubated overnight at room temperature in four volumes of 7
M CsCl. The next day, the samples were filtered through a 0.45-␮M
syringe filter and washed with TE buffer in a centricon 30 (Amicon,
Beverly, MA). An aliquot of each replication sample was analyzed on a 1%
agarose gel containing 5 ␮g/ml ethidium bromide. The gel was dried and
autoradiographed.

Mutant Analysis
Each replication reaction was digested with 0.025 U of DpnI (Boehr-
inger Mannheim, Indianapolis, IN), which specifically cuts input plasmids
that carry the E. coli dam-methylation pattern on both strands as opposed
to semiconservative replication products, which are hemimethylated or
unmethylated [Peden et al., 1980]. Reactions were then incubated with 3– 4
U dam methylase (New England Biolabs, Beverly, MA) to restore the
bacterial methylation pattern to newly synthesized strands. The efficiency
of each of the two enzymatic steps was confirmed by measuring transfor-
mation of plasmids from in vitro reactions with and without T-antigen.
Transformation was measured after incubation with DpnI and after redi-
gestion with DpnI of dam methylase-treated plasmid. The amount of each
enzyme used was based on titration experiments (data not shown).
The MBM7070 E. coli tester strain, F-, lacZamCA7020, lacY1, hsdR⫺,
hsdM⫹, araD139, ⌬(araABC-leu)7679, galU, galK, rpsL, thi [Hauser et
al., 1988], was transformed with the products of replication by using a
Cell-Porator apparatus (Bethesda Research Laboratories, Gaithersburg,
MD). The bacteria were grown on agar plates containing 50 ␮g/ml ampi-
cillin, spread with 5-bromo-4-chloro-3-indoly-␤-D-galactoside (X-gal, 2
mg/100-mm plate) and isopropyl-␤-D-thiogalactoside (IPTG,12 mg/
100-mm plate) [Seidman et al., 1985]. After restreaking, plasmid DNA
from white colonies was purified using a QIAprep spin kit (Qiagen). The
analysis of plasmids for altered DNA mobility representing gross alteration
was done by electrophoresis on 1% agarose gels containing 5 ␮g/ml
ethidium bromide.
The mutation spectra were collected from three independent experiment
dates. The plasmid DNA used in the first and second experiments differed
by one base pair at position 96 in the pre-tRNA region. We analyzed
published spectra generated from the plasmids pLS189(96C) or
pLS189(96G) [Levy et al., 1996a] using the statistical method described
below. They were not found to be significantly different (P ⫽ 0.2).
Therefore, mutant collection from the first and second experiments was
done in unison and the data were combined. In the first two experiments,
plasmid from most white colonies was analyzed for altered DNA mobility
on 1% agarose gels. In collecting mutants from the third experiment, with
pLS189(96C), plasmid from all white colonies was analyzed on 1% aga-
rose gels.
As a general rule, plasmids with altered mobility were omitted from
further analysis. As a result, it was possible that the first data set may have
included mutations from plasmids with altered mobility that were excluded
in the third experiment. To evaluate this possibility, the supF sequences of
26 plasmids with altered gel mobility were determined. Only 2/26 (7.7%)
contained a mutation in the supF gene. Because the possible contribution
of these mutations to the overall mutation spectra is small, the mutation
spectra for the same dose and extract type were combined and analyzed.
The analysis of altered gel mobility and sequenced large deletions, though,
remains exclusive to data from the third experiment.
Sequencing was completed by the University of Cincinnati DNA Core
using the Perkin–Elmer Biosystems Big Dye Terminator system with the
primer 5⬘-GGAAATGTTGAATACTC (New England Biolabs or Inte-
grated DNA Technologies, Coralville, IA). The distribution of mutations
from each reaction type was gathered by careful examination of computer-
generated signal profiles and recorded in the form of a mutation spectrum.
Western Immunoblotting
Samples were solubilized in Laemmli sample loading buffer, placed at
100°C for 3 min, and then separated on a 12% denaturing SDS–polyac-
rylamide gel. Proteins were transferred to Immobilon-P polyvinyl-divinyl
fluoride transfer (PVDF) membranes (Millipore, Bedford, MA) using a
semidry apparatus (Bio-Rad, Hercules, CA) at a maximum of 150 mA and
20 V for 1.5–2 hr. The membranes were blocked for 0.5–1 hr with TTBS
(100 mM Tris–HCl [pH 7.5], 0.9% NaCl, 0.3% Tween 20) containing 5%
dry milk and then probed with polyclonal anti-XPA at 1 ␮g/ml (sc-853;
XPA Protein and Mutagenesis 331
Santa Cruz Biotechnology, Santa Cruz, CA) for 1–2 hr. After washing four
times with TTBS, the membranes were incubated with donkey anti-rabbit
horseradish peroxidase-linked secondary antibody (1:10,000; Amersham
Life Science, Arlington Heights, IL). Membranes were washed three times
with TTBS and the proteins were visualized using the ECL method
(Amersham Life Science).
Colony-Forming Assay
LM217 or GM00637G cells (4000 – 60,000), or XP12BE-SV cells
(1500 – 40,000, depending on dose) were plated into 100-mm dishes and
grown for 1 day. The cells were irradiated with 0 –10 J/m2 UVC in
phosphate-buffered saline (Sigma, St. Louis, MO). After 14 days, colonies
were fixed with ethanol, stained with Giemsa, and counted. Figure 1
contains data from two separate experiment dates, with all cell lines
measured on each date. Each point represents two to six individual dishes.
Statistical Analysis
Mutational spectra were analyzed using an approximate Fisher’s exact
test for 2 ⫻ K tables [Adams et al., 1987]. The application of this test to
mutations in the supF gene is described elsewhere [Medvedovic et al.,
2001]. Multiple comparisons were performed based on the Fisher’s exact
test with resampling-based multiplicity adjustments [Westfall et al., 1989].
The test used to identify hotspots (sites having more mutations than
expected under the assumption of randomness) was previously described
by Tarone [1989]. For comparisons of the frequencies of mutants and
mutation types, the chi-square test for 2 ⫻ 2 tables was used. Values of
P ⬍ 0.05 were considered significant.
RESULTS
Characterization of the XP12BE-SV Cell Line
Colony-forming assays were performed to confirm the
UVC sensitivity of the XP-A cell line, XP12BE-SV, as
previously published [Tanaka et al., 1990]. Stocks of
XP12BE-SV, the control cell lines LM217 and GM00637G,
as well as XP12BE-SV cells newly received from the re-
pository, were tested. As shown in Figure 1, at a dose of 5
J/m2 UVC the survivals of LM217 and GM00637G cell
lines were approximately 35% of those of unirradiated
controls. At the same dose only 1–2% of irradiated
XP12BE-SV cells survived, demonstrating that the pub-
lished UVC-sensitive phenotype of this cell line was intact.
Survival of laboratory stocks closely matched that of newly
received cells.
The presence of the XPA protein in XP12BE-SV and
HeLa extracts was tested by Western immunoblotting using
a polyclonal antibody to the XPA protein. Figure 2 shows
that a protein of apparent molecular mass of 42 kDa oc-
curred in HeLa extract, but not in XP12BE-SV extract
(lanes 5 and 6). This band was also present as the strongest
band following immunoprecipitation of HeLa extract with
anti-XPA, but not in the supernatant of that reaction (lanes
2 and 3). The data suggest that this band (marked with an
arrow) is wildtype XPA protein; it is present in HeLa extract
but not present in XP12BE-SV extract. Immunoprecipita-
tion of HeLa extract with IgG yielded no bands, as ex-
332 King et al.
Fig. 2. Immunoblotting to test for the presence of the XPA protein using
an antipeptide antibody. The lanes contain 50 ng purified His-XPA (lane
1), immunoprecipitation (IP) of 200 ␮g HeLa extract with anti-XPA (lane
2), supernatant of HeLa IP (lane 3), 200 ␮g XP12BE-SV extract (lane 5),
or 200 ␮g HeLa extract (lane 6). Immunoprecipitation with IgG yielded no
bands (lane 4). XPA protein migrates with an apparent molecular mass of
42 kDa (lanes 2 and 6).
pected. Purified His-XPA runs as a higher molecular mass
band than does wildtype XPA protein, presumably because
of the additional amino acids of the histidine tag.
In Vitro Replication of UV-Irradiated pLS189 in
Extracts With or Without XPA Protein
The capacity of XP12BE-SV extracts to replicate irradi-
ated or unirradiated plasmid molecules was measured by an
in vitro replication assay. Plasmid irradiated with 40 J/m2
UVC or unirradiated was incubated in vitro with
XP12BE-SV extract, XP12BE-SV extract plus 25 ng XPA
protein, HeLa extract, or HeLa extract plus 25 ng XPA
protein under replication conditions (as described under
Materials and Methods). At various times aliquots of the
reactions were removed, incubated with PK/EDTA, and
spotted to filters, and the radioactive label incorporation was
measured. Irradiation of input plasmid with 40 J/m2 UVC
inhibited [␣-32P]dCTP incorporation in both HeLa and
XP12BE-SV extracts by an average of 63%, based on
measurements at the 3-hr time point (Fig. 3). Incorporation
tended to be slightly lower in reactions containing
XP12BE-SV extract as compared to those containing HeLa
extract (by 46% with unirradiated plasmid, 29% with irra-
diated plasmid). Because of scatter in the amount of incor-
poration in duplicate samples, however, it is unclear
whether the changes observed with addition of purified
XPA protein are biologically significant. In the absence of
T-antigen, little incorporation was observed in either extract
with unirradiated plasmid. Plasmid DNA purified from the
reactions was separated by agarose gel electrophoresis in
the presence of ethidium bromide and visualized by auto-
radiography. Labeled products appeared as form I (super-
coiled and relaxed closed circles), form II (nicked circles),
and an intermediate form presumed to contain circular plas-
mid dimers (Fig. 4).
Fig. 3. Incorporation of [␣-32P]dCTP into UV-irradiated or nonirradiated
pLS189 during DNA replication in vitro. Each point represents the total
incorporation level at specific times during a 3-hr reaction. Points are based
on two filter counts from a single reaction, or the average filter counts of
three to five reactions with standard errors of 7–37%. Lines: solid, reac-
tions with T-antigen; dashed, reactions without T-antigen. Symbols: open
symbols, XP12BE-SV extract; closed symbols, HeLa extract; F and E, 0
J/m2; Œ and ‚, 0 J/m2 ⫹ XPA protein; f and 䡺, 40 J/m2; 䉬 and 䉫, 40
J/m2 ⫹ XPA protein.
Mutant Frequency
The frequency of transformed bacteria containing repli-
cated plasmid with mutations in the supF gene was mea-
sured to determine whether XP12BE-SV extract has the
ability to fix mutations during replication of UV-irradiated
plasmid in vitro. Following incubation in replication condi-
tions, plasmid DNA from each reaction was digested with
DpnI to remove unreplicated molecules and with dam meth-
ylase to restore the bacterial methylation pattern prior to
transformation of E. coli. Plasmid isolated from white col-
onies was evaluated for altered mobility by agarose gel
electrophoresis and the supF region sequenced (as described
under Materials and Methods). Bacteria containing plas-
mids with base substitutions, deletions, or insertions of three
base pairs or less in the supF gene (positions 99 –183) were
scored as mutants. The total numbers of mutants and colo-
nies examined are shown in Table I. The spontaneous mu-
tant frequency following replication of unirradiated plasmid
was 0.010% in HeLa extract and 0.020% in XP12BE-SV
extract; a significant difference. The mutant frequency when
using plasmid irradiated with 40 J/m2 UVC increased by
14.7-fold to 0.147% in HeLa extract and by 11.7-fold to
0.233% in XP12BE-SV extract; the resulting frequencies
were also significantly different. Addition of 25 ng purified
XPA protein to reactions containing irradiated plasmid and
XP12BE-SV extract significantly lowered the mutant fre-
quency from 0.233 to 0.164%. Addition of XPA protein also
lowered mutation frequencies in reactions containing unir-
radiated plasmid. Addition of XPA protein to reactions
containing irradiated plasmid and HeLa extract had no
substantial effect on mutant frequency.

Fig. 4. Autoradiograph of 1% agarose gel containing the labeled products
of in vitro replication electrophoresed in the presence of ethidium bromide.
Plasmid pLS189 with a G at position 96 (A) or a C at position 96 (B) was
irradiated with 40 J/m2 UVC or unirradiated and replicated in HeLa or
XP12BE-SV (XP) extract in the presence or absence of T-antigen. Labeled
products predominantly appear as form I (supercoiled and relaxed closed
circular), form II (nicked circles), and an intermediate form presumed to
contain plasmid dimers.
Mutation Type
Mutations identified in the supF gene of replicated plas-
mids from each reaction condition were recorded according
to type. The frequencies of the predominant mutation types
are listed in Table I. Although the most frequent mutation
type in all UV-associated spectra was the GC 3 AT tran-
sition, the frequency of this change was decreased in XPA-
deficient extract. Of 160 total mutated sites identified in
UV-irradiated plasmids replicated in HeLa extract, 147
(91.9%) were GC 3 AT transitions. In contrast, replication
of UV-irradiated plasmid in XP12BE-SV extract produced
significantly fewer GC 3 AT transitions, 89 of 120 total
mutated sites observed (74.2%, P ⫽ 0.001). Addition of
purified XPA protein to XP12BE-SV extracts resulted in
GC 3 AT transitions at 56 of 59 total mutated sites, a level
20% higher than that with XP12BE-SV extract alone and
one similar to the HeLa spectrum. Each of the GC 3 AT
transitions occurred across from dipyrimidines, suggesting
XPA Protein and Mutagenesis 333
that they were the result of mutation fixation opposite UV-
induced pyrimidine dimers or 6-4 photoproducts. Tandem
double-base substitutions represented 3.2% of total muta-
tions in reactions containing irradiated plasmid and HeLa
extract, but were absent in reactions containing irradiated
plasmid and XP12BE-SV extract. Deletion mutations of one
or two base pairs represented a large percentage of muta-
tions observed in reactions with unirradiated plasmid, as
expected, but were also relatively common in reactions with
irradiated plasmid and XP12BE-SV extract. The 16.1%
deletion frequency in XP12BE-SV extract was over four-
fold higher than, and significantly different from, the 3.9%
observed in HeLa extract (P ⫽ 0.001). XP12BE-SV extract
with added XPA protein yielded 3.6% deletions, a value
similar to that of HeLa extract.
Sequence Specificity
The distributions of mutations resulting from in vitro
replication of UV-irradiated plasmid in XP12BE-SV or
HeLa extracts were recorded in the form of mutation spectra
(Fig. 5). The overall differences in spectra were evaluated
by statistical analysis, considering the total number of
changes and the number of changes at each site in the supF
gene. The distributions of mutations in HeLa 40 J/m2 and
XP12BE-SV 40 J/m2 spectra were significantly different
(P ⫽ 0.00006). The influence of addition of XPA protein on
mutation spectra was also examined. The HeLa 40 J/m2 and
HeLa 40 J/m2 ⫹ XPA spectra were significantly different
(P ⫽ 0.045). The XP12BE-SV 40 J/m2 and XP12BE-SV 40
J/m2 ⫹ XPA protein spectra were significantly different in
analyses excluding the large number of deletion mutations
present in the XP12BE-SV 40 J/m2 spectrum (P ⫽ 0.009 for
base substitutions, P ⫽ 0.12 for all mutations). The two
spectra from reactions in which XPA protein was added,
HeLa 40 J/m2 ⫹ XPA and XP12BE-SV 40 J/m2 ⫹ XPA,
were also significantly different from each other (P ⫽
0.012). The prediction that addition of XPA protein to
XP12BE-SV extract would restore the spectrum to that of
HeLa extract was also tested. Indeed, the XP12BE-SV 40
J/m2 ⫹ XPA spectrum was not significantly different from
that of HeLa 40 J/m2 (P ⫽ 0.14).
Hotspots of mutagenesis were identified in each of the
spectra generated from replication of UV-irradiated plas-
mid, as described previously [Tarone, 1989]. Based on
published results of replication of UV-irradiated plasmids
using extracts [Carty et al., 1995] or cells [Bredberg et al.,
1986; Levy et al., 1996a], hotspots at positions 99, 100, 155,
and 156 of the supF gene represent the characteristic UV-
induced mutation pattern. In our data, positions 100, 108,
and 156 were hotspots in the HeLa 40 J/m2 spectrum. Our
XP12BE-SV 40 J/m2 spectrum did not display hotspots at
positions 100, 108, or 156, but instead contained hotspots at
positions 124, 133, and 164. The XP12BE-SV 40 J/m2 ⫹
XPA protein spectrum retained the hotspot at position 164,
334 King et al.

TABLE I. Frequencies of SupF Mutants and Mutation Types

Cell extract: HeLa XP12BE-SV HeLa XP12BE-SV HeLa XP12BE-SV HeLa XP12BE-SV
UVC: 0 J/m2 0 J/m2 0 J/m2 0 J/m2 40 J/m2 40 J/m2 40 J/m2 40 J/m2
XPA protein: ⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹

Total colonies 297,162 111,745 43,776 25,011 105,260 49,798 37,890 33,590
Total mutantsa 31 22 3 2 155 116 53 55
Mutant frequency (%) 0.010 0.020 0.007 0.008 0.147 0.233 0.140 0.164
Tandem base substitutions (%)b 0.0 4.2 — — 3.2 0.0 1.9 5.3
Deletions (%)c 39.4 16.7 — — 3.9 16.1 0.0 3.6
GC 3 AT transitions (%)d 39.4 55.6 — — 91.9 74.2 96.3 94.9
a
Mutants are bacteria containing plasmid with a sequenced base substitution, deletion, or insertion of three base pairs or less.
b
Tandem base substitutions are the percentage of total mutations.
c
Deletions are the percentage of total mutations.
d
GC 3 AT transitions are the percentage of total mutated sites.

but did not display hotspots at positions 124 or 133. The
XP12BE-SV 40 J/m2 ⫹ XPA protein spectrum also con-
tained a hotspot at position 178.
Apart from determining the location of statistically sig-
nificant hotspots within individual spectra, we also used
those sites as the basis to look for trends in the mutation
frequencies at specific sites between spectra. The mutation
frequency at positions 99, 100, 155, and 156 in the HeLa 40
J/m2 spectrum was 26.5% of all mutations compared to
12.7% in the XP12BE-SV 40 J/m2 spectrum (Table II). This
is a 2.1-fold higher mutation frequency at these sites in
extract containing the XPA protein compared to those in
XPA-deficient extract. The difference increased to threefold
when only base substitutions were analyzed. Addition of
XPA protein to reactions containing XP12BE-SV extract
produced a more HeLa-like spectrum, approximately dou-
bling the frequency of mutagenesis at positions 99, 100,
155, and 156 (25.0% of all mutations). The mutation fre-
quencies at positions 124 and 164 were not strikingly dif-
ferent between extract types and are also presented in Table
II. The mutation frequency at position 133 in the HeLa 40
J/m2 spectrum was only 3.2% compared to 10.2% in the
XP12BE-SV 40 J/m2 spectrum, a 3.2-fold increase in XPA-
deficient extract. This difference increased to 3.6-fold when
only base substitutions were analyzed. Addition of the XPA
protein to reactions containing XP12BE-SV extract produced a
more HeLa-like spectrum, reducing the mutation frequency at
position 133 from 10.2 to 7.1% of all mutations.
Gel Mobility and Large Sequenced Deletions
During the collection of the mutation data, several white
colonies yielded plasmids with no mutations in the supF
gene, as determined by sequencing. To characterize these
plasmids, all plasmids from white colonies from one of
three experiment dates were tested for altered mobility by
agarose gel electrophoresis. A total of 107 white colonies
were identified from reactions containing UV-irradiated
plasmid and HeLa extract. Of these, 8 failed to yield plas-
mid after at least two preparations, 30 contained plasmid
with altered mobility, 20 contained plasmids of expected
size but with no mutations in supF, 1 contained plasmid
with a mutation outside the sequence of supF, and 2 con-
tained plasmid with a large sequenced deletion. Only 46 of
107 white colonies actually contributed to the spectrum.
The results of the 93 white colonies identified from reac-
tions containing UV-irradiated plasmid and XP12BE-SV
extract were similar. They included 4 that failed to yield
plasmid, 28 that contained plasmid with altered mobility, 11
that contained plasmid of expected size but with no muta-
tions in supF, 1 that contained plasmid with a large se-
quenced deletion, and 49 contributed to the spectrum. The
frequency of altered gel mobility, sequenced deletions
larger than three base pairs, and sequenced mutations three
base pairs or less are presented in Table III. Altered gel
mobility did not correlate with the presence of the XPA
protein, although altered mobility did correlate with use of
UV-irradiated plasmid. The frequency of deletions larger
than three base pairs was small, but also increased in rep-
lication reactions containing UV-irradiated plasmid.
DISCUSSION
We have demonstrated the capacity of extracts made
from HeLa cells or from an XPA-deficient cell line,
XP12BE-SV, to replicate UV-irradiated or unirradiated
plasmid DNA in vitro. Although UV-irradiation of input
plasmid inhibited replication by an average of 63%, com-
pletely replicated molecules containing fixed mutations op-
posite sites of dipyrimidines were isolated. In comparison to
HeLa extract, XP12BE-SV extract had a slightly lower
ability to replicate either UV-irradiated or unirradiated plas-
mid, and this difference appeared to diminish by addition of
purified XPA protein. The forms of replicated product DNA
were the same in both extract types.
We have shown that mutant frequencies and mutation
types resulting from replication of UV-irradiated plasmid in
HeLa or XP12BE-SV cellular extracts differ in several
 XPA Protein and Mutagenesis 335

Fig. 5. Distributions of mutations in the supF gene obtained from replication of UV-irradiated or unirradiated plasmid in HeLa or XP12BE-SV extract,
with or without added XPA protein. Boxes represent deletions, underlined bases represent mutations occurring as multiples in individual plasmids, ^
denotes an insertion, stars appear above statistically significant hotspots, and columns represent sites characteristic of UV-induced mutagenesis.

ways. First, the mutant frequency of bacteria transformed
with replicated plasmid was higher from reactions contain-
ing XP12BE-SV extract than from those containing HeLa
extract. The difference in mutation frequency between UV-
irradiated plasmid replicated in HeLa or XP12BE-SV ex-
tracts was diminished by addition of purified XPA protein to
XP12BE-SV extract. The second major difference between
HeLa and XP12BE-SV extracts was in the types of muta-
tions observed. Replication of UV-irradiated plasmid in
HeLa extract resulted in a predominance of GC 3 AT
transitions (91.9%). This agrees with published results in
HeLa extract or XP12BE-SV cells, showing GC 3 AT
mutations at frequencies of 87.0 –94.3% (Table IV, columns
B, F–I). Replication of UV-irradiated plasmid in
XP12BE-SV extract resulted in a significantly lower fre-
quency of GC 3 AT transitions (74.2%), which increased
to a HeLa-like level in reactions containing XP12BE-SV
extract and XPA protein (94.9%). These results suggest that
the decrease in GC 3 AT transition mutations is specific to
replication in XPA-deficient extracts.
336 King et al.

TABLE II. Frequencies of Mutations at Specific Sites in the SupF Gene

Cell extract: HeLa XP12BE-SV HeLa XP12BE-SV
UVC: 40 J/m2 40 J/m2 40 J/m2 40 J/m2
XPA protein: ⫺ ⫺ ⫹ ⫹

Frequency of all mutations

Mutations at 99, 100, 155, or 156 (%) 26.5 12.7 18.9 25.0
Mutations at 124 (%) 4.5 6.8 7.5 3.6
Mutations at 133 (%) 3.2 10.2 5.7 7.1
Mutations at 164 (%) 5.2 9.3 7.5 10.7
Frequency of base substitutions
Substitutions at 99, 100, 155, or 156 (%) 27.5 9.1 18.9 25.9
Substitutions at 124 (%) 4.0 8.1 7.5 3.7
Substitutions at 133 (%) 3.4 12.1 5.7 7.4
Substitutions at 164 (%) 5.4 11.1 7.5 11.1

TABLE III. Characteristics of Plasmids Isolated From White Colonies

Mutation frequency (%)
Altered gel
mobility Sequenced Sequenced
Total colonies (%) large deletions other

0 J/m2 UVC
HeLa extract 56,317 0.012 0.000 0.002
HeLa extract ⫹ XPA protein 43,776 0.009 0.000 0.007
XP12BE-SV extract 46,410 0.006 0.004 0.011
XP12BE-SV extract ⫹ XPA protein 25,011 0.000 0.000 0.008

40 J/m2 UVC
HeLa extract 34,719 0.086 0.006 0.132
HeLa extract ⫹ XPA protein 37,890 0.071 0.003 0.140
XP12BE-SV extract 24,798 0.113 0.004 0.198
XP12BE-SV extract ⫹ XPA protein 33,590 0.063 0.003 0.164

Another characteristic specific to replication of UV-irra-
diated plasmid in XP12BE-SV extract was a significant
increase in deletion mutations to 16.1%, over a fourfold
increase compared to that of HeLa extract. Because dele-
tions may be a more harmful type of mutation, possibly
leading to changes in the reading frame, this result suggests
that the presence of the XPA protein is a positive factor in
the way cells respond to DNA damage during replication.
The third type of mutation, which differed in frequency
between replication in HeLa and that in XP12BE-SV ex-
tract, was the tandem double-base substitution (TDS). Stud-
ies using pZ189 have observed TDS at a frequency of
11.1–20.0% (Table IV, columns A–C, F). In the present
study, TDS accounted for only 3.2% of total mutations in
HeLa extract and 0.0% in XP12BE-SV extract. In conjunc-
tion with published data showing the frequency of TDS
following replication of UV-irradiated pLS189 in
XP12BE-SV cells at 2.3–7.2% (Table IV, columns G and
H), the data suggest that low TDS frequencies correlate with
use of the pLS189 plasmid construct, not with a particular
extract or method of replication.
Analysis of the location of mutations in the supF gene of
replicated plasmids revealed a nonrandom pattern of mu-
tagenesis that differed significantly between HeLa and
XP12BE-SV extracts. Replication of UV-irradiated plasmid
in HeLa extract resulted in 26.5% of total mutations occur-
ring at positions 99, 100, 155, and 156, sites characteristic
of UV-induced mutagenesis. Positions 100 and 108 were
statistically significant hotspots, as observed in UV mu-
tagenesis studies of pLS189 replicated in XP12BE-SV cells
[Levy et al., 1996a]. Position 156 was also a hotspot, as seen
following replication of UV-irradiated pZ189 in HeLa ex-
tract [Carty et al., 1995] and in CV-1, XP12BE-SV, or
GM00637 cells [Bredberg et al., 1986; Hauser et al., 1986;
Levy et al., 1996a]. In contrast, the spectrum of mutations
produced by replication of UV-irradiated plasmid in
XP12BE-SV extract produced only 12.7% of total muta-
tions at positions 99, 100, 155, and 156 and did not yield
hotspots at positions 100, 108, or 156. Instead, positions
124, 133, and 164 were determined to be hotspots in the
XP12BE-SV extract. These hotspots are not plasmid con-
struct specific, because pLS189 replicated in XP12BE-SV
cells did not display them [Levy et al., 1996a]. The differ-
ences that we observed in the hotspots identified in HeLa or
XP12BE-SV spectra do not correlate with differences in the
published frequencies of UV-induced pyrimidine dimers or
6-4 photoproducts at specific sites in the supF gene [Brash
et al., 1987]. Also, based on the UV-induced mutation
 XPA Protein and Mutagenesis 337

1.2c

The data in columns A–J correspond to the following references: A, Hauser et al., 1986; B, Carty et al., 1995; C and F, Bredberg et al., 1986; D and J, Levy et al., 1995; E and I, Myrand et al., 1996;
spectra from GM0637 and XPA-deficient XP2OS cells,

15.2

94.6

5.1
7.6
7.6
J

100–1000
pSP189
XP2OS

NAf
In vivo
Levy et al. [1995] concluded that mutation hotspots do not
correlate with the DNA repair status of the cell line.
Addition of XPA protein to HeLa cell extract already
containing wildtype XPA protein significantly altered the

The frequency of mutations at positions 99, 100, 155, and 156 of the supF gene is not applicable because of the use of a plasmid that differs in sequence from pLS189 at position 99.
XP12BE-SV

spectrum of mutations and eliminated the characteristic

1.7c
19.3

91.2

21.1
0.0
1.8
NAf
pSP189
In vivo
I

100

UV-induced hotspot at position 156, while increasing the

percentage of mutations at other sites, including positions
124, 133, and 164 (Fig. 5, Table II). A similar change in
mutation spectrum was reported when XPA-deficient cells,
pLS189(96G)
XP12BE-SV

XP2OS, were transfected with XPA cDNA expressing XPA

2.3

94.3
41.9
1.2
1.2
5.8
NAd
H

300

protein at a level 14-fold over that of GM0637 cells [Levy

When unavailable in spectra, deletions and tandem base substitution frequencies are the percentage of total mutated plasmids as described in published tables.
In vivo

et al., 1995]. The authors reported that no significant dif-

ferences at specific sites were observed between the spectra
of GM0637, XP2OS, and XPA-overexpressing XP2OS
pLS189(96C)
XP12BE-SV

cells, other than an increase at position 124 and a decrease

7.2

89.1
25.6
2.7
4.0
2.7
NAd

at position 156 in the XPA-overexpressing cells. These data

G

300
In vivo

The data in this table are derived from published mutation spectra. Frequencies are the percentage of total mutations, except where noted.

indicate that changes in XPA protein stoichiometry alter the

specificity of UV-induced mutagenesis, perhaps as a result
of changes in protein–protein interactions. Based on data
XP12BE-SV

from that study, using our method of analysis, we deter-

20.0
1.7
93.1

3.3
0.0
0.0
F

50–300

NAf

mined that the mutation spectra generated in XP2OS or

In vivo
pZ189

GM00637 cells are not significantly different (P ⫽ 0.068).

In addition, the XP2OS cells yielded a hotspot at position
156, a characteristic UV-induced site. These data indicate
GM00637

that the mutation spectra of UV-irradiated plasmid repli-

3.9c
pSP189

6.1

80.8

4.1
2.0
4.1
NAf
In vivo

cated in vivo, in XPA-proficient or -deficient cell lines, do

E

100

not correlate with the repair status of the cell lines. This is
in agreement with the in vivo spectra of other XPA-deficient
cell lines (Table IV, columns F–I).
3.8c
GM00637

11.4

78.5

1.4
1.4
5.7
D

300–1000
pSP189

NAf
In vivo

Our data suggest that the XPA protein influences the

specificity of UV-induced mutagenesis during replication in
vitro. Results from the addition of purified XPA protein to
TABLE IV. Characteristics of Mutations Induced by UV-Irradiationa

replication reactions support this conclusion. As expected,

5.6c
GM00637

11.1

72.8

1.1
2.2
0.0

addition of XPA to reactions containing XP12BE-SV ex-

GC 3 AT transition frequencies are the percentage of total mutated sites.
C

100–1000

NAf
In vivo
pZ189

tract altered several of the characteristics of XP12BE-SV

extract and yielded a more HeLa-like spectrum. Those
changes included significantly decreasing the mutant fre-
In vitro

quency and frequency of deletion mutations, as well as

pZ189

15.0
7.5
87.0

0.0
0.0
0.0
NAf
HeLa
B

40

alleviating the hotspots at positions 124 and 133. Addition

of XPA also increased the frequency of mutations at posi-
tions 99, 100, 155, and 156. These observations demonstrate
In vivo

16.7c
3.1c
pZ189

61.6

3.6
1.4
2.2
NAf
CV-1

that the differences in spectra were the result of loss of a

A

500

function of the XPA protein.

The spectrum of mutations generated in XP12BE-SV
Some information was not available.
Referenceb:
Cell type:
Replication system:
Plasmid:
UV dose (J/m2):

Mutations at 99, 100, 155, 156 (%)

extract is novel, neither like the spectrum generated in the

same type of cellular extract made from HeLa cells [Carty
Tandem base substitutions (%)

G and H, Levy et al., 1996a.

et al., 1995] nor the in vivo replication results of the same

GC 3 AT transitions (%)e

plasmid replicated in the XP12BE-SV cell line in culture

Mutations at 124 (%)
Mutations at 133 (%)
Mutations at 164 (%)

[Levy et al., 1996a]. We propose that the novel spectrum

generated in XP12BE-SV extract is attributed to the absence
Deletions (%)

of functional XPA protein combined with the loss of a

nuclear protein, with overlapping function, which is perhaps
retained in the nuclear pellet during cellular extract prepa-
ration. The published spectra are consistent with this hy-
b

d
a

f
338 King et al.
pothesis, suggesting that HeLa extract contains functional
XPA protein, whereas cultured XP12BE-SV cells contain
the nuclear protein with overlapping function. Given that
proteins with overlapping functions are common features of
cellular processes, the possibility of additional proteins in-
volved in no way lessens our in vitro observation of a novel
function for XPA in modulating mutation fixation during
replication of UV-irradiated DNA.
Several mechanisms have been proposed to explain how
the DNA replication machinery responds to UV-induced
damage sites. Results of replication of UV-damaged viral
DNA in monkey cells [Sarasin and Hanawalt, 1980] and in
cellular extracts [Svoboda and Vos, 1995] suggest that
lesions block replication when located in the leading strand,
while lagging strand synthesis continues to occur. A single-
stranded region of unknown length forms across from the
lesion site. When the lesion is located in the lagging strand,
a pause in replication may occur. However, synthesis of the
next Okazaki fragment would continue and result in a small,
single-stranded region across from the lesion site. Studies
with purified proteins from E. coli or S. cerevisiae, as well
as cloning of polymerase ␩ [Masutani et al., 1999], agree
with a proposed model of translesion synthesis [Woodgate,
1999]. In this model, the normal pol ␦– containing complex
dissociates from the damaged strand and is replaced by a
lesion-replicating polymerase complex. Once one or two
nucleotides are synthesized, the lesion-replicating polymer-
ase complex disassociates. The normal polymerase complex
then reassembles and resumes replication.
Our data suggest that the XPA protein is a modifier of
translesion replication. The higher mutation frequency and
altered hotspot pattern observed following replication of
UV-irradiated plasmid in XPA-deficient extract, as well as
data with purified XPA, suggest that the replication com-
plex responds to the presence of the XPA protein. XPA is
not a polymerase, nor is it required for DNA replication or
even translesion replication. Nonetheless, the inherent ac-
tivities of the XPA protein, as well as its actions during
NER, correlate with a function during translesion replica-
tion. The XPA protein preferentially binds to undamaged
ssDNA vs. dsDNA [Jones and Wood, 1993], and to UV-
damaged vs. undamaged dsDNA [Robbins et al., 1991;
Wakasugi and Sancar, 1998]. Binding to UV-damaged
DNA is mainly the result of 6-4 photoproducts [Jones and
Wood, 1993]. The ability of XPA to bind to UV-damaged
DNA is greatly increased by association with RPA [He et
al., 1995]. The XPA protein contains a zinc-finger domain
associated with DNA-binding ability [Tanaka et al., 1990;
Asahina et al., 1994]. In its repair-associated role, XPA is
required during the damage-recognition step of NER
[Wakasugi and Sancar, 1998]. It binds to the lesion as a
complex with RPA. Following lesion recognition and bind-
ing, XPA recruits and binds several additional proteins
through individual binding domains. During replication of
damaged DNA, we propose that uncoupling of fork pro-
gression creates an area of lesion-containing ssDNA to
which XPA binds. Although it is unknown how XPA bind-
ing affects translesion replication, it may alter fork progres-
sion to allow more time for lesion-replicating polymerases
to be recruited. The likelihood of recruiting a more or less
error-prone polymerase may be affected by the interactions
of XPA with surrounding proteins. It is also not unreason-
able to suppose that sequence context is important because
we know that single nucleotide changes have proximal and
distal effects on mutation fixation during replication of
UV-damaged plasmid DNA in vivo [Levy et al., 1996b].
Additional published information may be consistent with
our observations. The homolog of the XPA protein in E.
coli, UvrA, inhibits translesion replication through an abasic
site analog on a 40-bp oligo by purified polymerases I, II, or
III. UV-induced His⫺ to His⫹ reversion mutations are
3.3-fold higher in a bacterial strain lacking both UvrA and
DNA photolyase. The researchers suggested that UvrA, the
S. cerevisiae homolog Rad 14, and XPA may each directly
inhibit translesion synthesis at UV-induced damage sites,
thereby decreasing mutagenesis by preventing likely mis-
coding events [Paz-Elizur et al., 1997]. This agrees with our
data, in which HeLa extract showed a lower mutation fre-
quency than that of XP12BE-SV extract. In another study
related to the XPA protein, the p53 mutation spectrum of
UVB-induced skin tumors of XPA-deficient mice did not
display the hotspots of mutagenesis observed in the p53
genes of UVB-exposed wildtype mice [Takeuchi et al.,
1998]. Although comparison to a spectrum generated in
another NER-deficient transgenic mouse would be valuable,
this study may also support the role of the XPA protein in
influencing the specificity of UV-induced mutagenesis.
Our work is the first to examine the role of the XPA
protein in UV-induced mutagenesis during DNA replication
in vitro. Although others have reported differences in UV-
induced mutation spectra generated in NER-proficient or
-deficient cultured cells [Bredberg et al., 1986], our obser-
vations using cellular extracts suggest a novel function of
XPA in UV-induced mutagenesis by modulating translesion
replication, apart from its previously characterized role in
repair of UV-damaged DNA. Using purified XPA protein,
we have shown that XPA alters the products of in vitro
replication of UV-irradiated plasmid such that significantly
different mutation spectra are generated. Because mutations
at some locations, such as within tumor suppressors and
cellular oncogenes, are more likely to lead to cancer, we
believe that the XPA protein is an important factor in the
link between mutagenesis and carcinogenesis.
ACKNOWLEDGMENTS
We thank Michael Seidman for plasmid pLS189,
XP12BE-SV cells, and helpful discussions. We are grateful
to Aziz Sancar for purified XPA protein, Leon Kapp for
LM217 cells, and Michael Carty for many helpful discus-

sions. This work was supported by NIH grant ES05400 to
K. D., by NIEHS Center grant ES06096, and by NIH
training grant ES07250.
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Accepted by—
T. A. Cebula