MOLECULAR AND CELLULAR BIOLOGY, Jan. 1994, p. 68-76
Copyright © 1994, American Society for Microbiology
An Interaction between the Mammalian DNA Repair Protein
XRCC1 and DNA Ligase III
KEITH W. CALDECOTT,1 CATHERINE K. McKEOWN,1 JAMES D. TUCKER,1 SIV LJUNGQUIST,2
AND LARRY H. THOMPSONl*
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore,
California 94551-0808,1 and Institute ofEnvironmental Medicine, Karolinska Institute,
S-10401 Stockholm, Sweden2
Received 12 July 1993/Returned for modification 9 August 1993/Accepted 26 September 1993
XRCCI, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a
protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating
agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was
constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX,
fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not
detrimental to XRCC1 activity. Affinity chromatography ofextract from EM9 cells transfected with pcD2EHX
resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA
ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with
pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected
EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two
proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels
in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or
pcD2EX. These findings indicate that XRCC1 is required for normal levels ofDNA ligase IIIactivity, and they
implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.
The Chinese hamster ovary (CHO) cell mutant EM9 is
hypersensitive to ethyl methanesulfonate (EMS) (10-fold)
and ionizing radiation (1.8-fold), and it is unable to grow in
medium containing chlorodeoxyuridine (CldUrd) under con-
ditions in which 20% of genomic Thy is replaced by chloro-
uracil during DNA replication (9, 29). EM9 repairs -y-ray and
EMS-induced single-strand breaks at a reduced rate and
exhibits a 10-fold increase in the occurrence of sister chro-
matid exchange (SCE) (27, 28). The sensitivity of EM9 to
alkylating agents is suggestive of a defect in the base
excision repair pathway, which involves sequential action by
DNA glycosylase, apurinic-apyrimidinic endonuclease, de-
oxyribose-phosphodiesterase, DNA polymerase, and DNA
ligase activities (17). The reduced rate of single-strand break
rejoining suggests that the defect in EM9 lies within a
postincision step of this pathway. EM9 is phenotypically
similar to cells derived from individuals with Bloom's syn-
drome (BS), a cancer-prone autosomal recessive disorder
characterized by high SCEs (10-fold) and sensitivity to
alkylating agents (4, 14, 15). Although chromosome localiza-
tion and somatic cell hybrid complementation studies indi-
cate that the EM9 and BS defects are genetically distinct (18,
21, 23), the two gene products most likely function in the
same, or a closely related, biochemical pathway. The phe-
notype of both EM9 and BS cells might be explained by a
defect in one or more of the three DNA ligases so far
identified in mammalian cells, which are designated DNA
ligases I, II, and III, respectively (24, 31). DNA ligase I plays
a major role in DNA replication but also appears to be
*Corresponding author. Mailing address: BBR Program, L452,
Lawrence Livermore National Laboratory, P.O. Box 808, Liver-
more, CA 94551-0808. Phone: (510) 422-5658. Fax: (510) 422-2282.
Electronic mail address: Larry_Thompson.bio MZ@biomed.llnl.
involved in the repair ofDNA damage caused by a variety of
agents, including alkylating agents (reference 1 and refer-
ences therein), and DNA ligase II has been proposed to play
a role both in DNA repair and DNA recombination (31). As
yet, no specific role has been identified for DNA ligase III
(31). Altered DNA ligase activity in BS cells has been
observed in several studies, in which it was proposed that
the activity ofDNA ligase I was affected (5, 6, 33, 34), but no
major abnormality in DNA ligase activity was found in the
one reported study in which EM9 was examined (7). How-
ever, only DNA ligase II activity was conclusively shown to
be normal in EM9 in this study, since DNA ligase I and III
activities were not distinguished from each other. Thus, it is
possible that a specific defect in one of these enzymes was
masked by the combined level of DNA ligase I and III
activities that was measured in the study.
The human gene that corrects the repair defect in EM9 has
been cloned and is designatedXRCCI (28). The gene is 33 kb
in length and encodes a 2.2-kb transcript and a correspond-
ing protein of 633 amino acids (69.5 kDa). To isolate XRCC1
protein and address its biochemical role in DNA strand
break repair, fully functionalXRCCI cDNA minigenes have
been constructed to allow overexpression of XRCC1 in
mammalian and prokaryotic cells (3). In this work, we
describe the expression of oligohistidine-tagged XRCC1 in
EM9 cells and present results suggesting that a DNA ligase
activity is indeed affected in EM9. This study demonstrates
that XRCC1 is required for normal levels of DNA ligase III
activity and strongly suggests that these two proteins are
physically associated within the cell.
MATERIALS AND METHODS
Cell lines and culture conditions. Isolation of the XRCC1
CHO mutants EM9 and EM-Cll, as well as the conditions
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DNA REPAIR PROTEINS XRCCI AND DNA LIGASE III
used for the routine propagation of these cell lines and their
transfection derivatives, have been described elsewhere (27,
28, 37). Eschenchia coli XL1-Blue (Stratagene, La Jolla,
Calif.) was used for routine maintenance and manipulation of
plasmids; the expression hosts for pET16BX were E. coli
HMS174(DE3) and BL21(DE3) (Novagen, Madison, Wis.)
(25, 26). The DE3 lysogen present in these cell lines encodes
T7 RNA polymerase under the control of thePtacinducible
(by isopropylthiogalactopyranoside [IPTG]) promoter. Un-
less stated otherwise, E. coli strains were grown in Luria-
Bertani (LB) medium at 37°C (22).
XRCC1 expression constructs and oligonucleotides. The
mammalian expression construct pcD2EHX contains the
full-length XRCC1 open reading frame (ORF) and was con-
structed as described previously for pcD2EX, using a pair of
complementary oligonucleotides to complete the coding
region at the 5' end (3). pcD2EHX differs from pcD2EX only
by the presence of an additional 18 bp that are located near
the 5' terminus of the XRCCI ORF and which encode a
hexahistidine tag to facilitate affinity purification (Fig. 1).
pcD2EHCX was constructed in the same way, but by using
a pair of oligonucleotides that contained an additional 27 bp
located immediately downstream of the histidine tag. The
27-bp region encodes a well-defined nine-amino-acid epitope
from the influenza virus hemagglutinin protein, which is
recognized by monoclonal antibody 12CA5 (35). The com-
plementary oligonucleotides used to construct pcD2EHX
and pcD2EHCX were generated on an automated synthe-
sizer and were purified with NENSORB cartridges as di-
rected by the manufacturer (DuPont/NEN Research Prod-
ucts, Boston, Mass.).
The prokaryotic expression construct pET16BX was gen-
erated by ligating an oligonucleotide duplex encoding the
first 37 bp of theXRCCI ORF to a truncated cDNA fragment
encoding the remainder of theXRCC1 ORF and by inserting
the resulting fragment into the NcoI site (ATG cloning site)
of the expression vector pET16b (Novagen). No additional
amino acids are present in the XRCC1 protein expressed
from this construct (Fig. 1). For expression of XRCC1 from
pET16BX, 10- to 20-ml starter cultures containing ampicillin
(0.1 mg/ml) were inoculated with HMS174(DE3) or BL21
(DE3) cells harboring pET16BX (from a fresh colony or
directly from frozen stocks), grown to mid-log phase (optical
density at 600 nm of 0.6 to 1.0), and placed at 4°C overnight.
Cells from the starter culture were pelleted and resuspended
in an equal volume of medium (plus ampicillin), and aliquots
were used to inoculate 0.1- to 1-liter cultures (plus ampicil-
lin) at a 1:100 dilution. XRCC1 expression was induced in
mid-log phase cells (optical density of -0.6) by the addition
of IPTG to 1 mM.
For specific radiolabeling of XRCC1, BL21(DE3) cells
harboring pET16BX were cultured as described above (with
the exception that M9 broth  was used instead of LB) and
were treated with rifampin (Sigma, St. Louis, Mo.) at 0.2
mg/ml 30 min after induction to inhibit E. coli RNA poly-
merase, thus allowing selective expression of XRCC1 from
the T7 promoter present in pET16BX (25, 26). Cell samples
(0.5 ml) were subsequently pulse-labeled for 15 min with 5
,uCi of [35S]methionine (1,000 Ci/mmol, 10 mCi/ml; Amer-
sham Corp., Chicago, Ill.) at the times indicated. Pelleted
cells from these samples were frozen at -20°C until needed,
when they were lysed by the addition of hot sodium dodecyl
sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)
loading buffer, and aliquots were subjected to electrophore-
sis through SDS-7.5% polyacrylamide gels. Gels were fixed
in 10% acetic acid for 10 min, stained with Coomassie blue,
AAMCCACGACGTTGAC ATG CCG GAG ATC CGC CTC CGC CATGTC GTG TCC TGCA
GGTGCTGCAACTG TAC GGCCTC TAG GCG GAG GCG GTA CAG CACAGG
MET PRO GLU ILE ARG LEU ARG HIS VAL VAL SER CYS
HIS HIS HIS HIS HIS HIS
HIS HIS HIS HIS HIS HIS TYR PRO TYR ASP VAL PRO ASP TYR ALA
C ATG COG GAG ATC CGC CTC CGC CATGTC GTG TCC TGCA
GGC CTC TAG GCG GAG GCG GTA CAG CAC AGG
MET PRO GLU ILE ARG LEU ARG HIS VAL VAL SER CYS
FIG. 1. Schematic showing the construction of the XRCC1 ex-
pression constructs used in this study. (A) The oligonucleotide
duplex used previously to reconstruct the full-length XRCCI ORF
present in pcD2EX. The upper oligonucleotide encodes 17 bases of
leader sequence and the first 37 bases of the ORF (italics). The
17-base leader sequence differs from the native XRCCJ leader
sequence by two bases at the 5' end of the oligonucleotide, where
GpC was replaced by TpT in the oligonucleotide to generate an
EcoRI terminus (underlined). (B) A second oligonucleotide duplex
was produced that in addition encoded a hexahistidine region
situated after the third amino acid. The oligonucleotide duplexwas
ligated to the 2.15-kb PstI-EcoRI fragment shown inpanel A,which
spans the remainder of the XRCC1 ORF, and the resulting EcoRI
XRCC1 cDNA cassette was inserted into the EcoRI site of the
expression vector pcD2E to generate the expression construct
pcD2EHX. (C) pcD2EHCX was constructed as described above,
using an oligonucleotide duplex that additionally encoded the
epitope recognized by monoclonal antibody 12CA5 (underlined). (D)
The oligonucleotide duplex shown, which encodes the first 37bpof
the XRCCI ORF, was ligated essentially to the XRCC1 PstI-EcoRI
fragment shown in panel A (but in which the EcoRI terminus was
converted to a blunt end with Klenow fragment), and theresulting
fragment (NcoI-blunt) was inserted into the bacterial expression
vector pET16b at the ATG cloning site to generate the XRCC1
expression construct pET16BX.
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CALDECOTT ET AL.
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