Sensitivity of group F xeroderma pigmentosum cells to UV and mitomycin C relative to levels of XPF and ERCC1 overexpression. Takashi Yagi0, Akiko Katsuya, ...
Mutagenesb vol.13 no.6 pp.595-599, 1998
Sensitivity of group F xeroderma pigmentosum cells to UV and mitomycin C relative to levels of XPF and ERCC1 overexpression
Takashi Yagi0, Akiko Katsuya, Akiko Koyano and Hiraku Takebe Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshida-konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
Introduction Patients with the cancer-prone hereditary disease xeroderma pigmentosum (XP) are classified into seven genetic complementation groups (A-G) of nucleotide excision repair (NER)deficient type and one variant of NER-proficient type (Kraemer et al., 1987). Rodent DNA repair-deficient mutant cells have been classified into 11 genetic complementation groups (excision repair cross-complementing; ERCC1-11) (Hoeijmakers, 1993). Genes correcting the defect of most of these complementation groups have been cloned and the defect in XP-F and ERCC-4 cells was found to be corrected by an identical gene (Brookman et al., 1996; Sijbers et al., 1996a). The XPF (ERCC4) gene encodes a 103 kDa protein, a human homolog of the budding yeast Radl protein (Brookman et al., 1996; Sijbers et al., 1996a). The XPF protein is barely detected in all XP-F cells examined, but all XP-F cells have a low level of nucleotide excision repair ability and intermediate UV sensitivity (Yagi et al., 1997). The yeast Radl protein makes a tight complex with RadlO protein and has a structure-specific endonuclease activity (Bardwell et al., 1994; Davies et al., 1995). Like the yeast Radl-RadlO complex, the human XPF protein tightly binds to ERCC1 protein, which is a yeast RadlO homologue (Park and Sancar, 1994; Aboussekhra et al., 1995; Matsunaga et al., 1996; Sijbers et al., 1996a). The XPF-ERCC1 complex and XPG proteins incise on the 5'- and 3'-sides of pyrimidine dimers respectively in DNA of UV-irradiated cells and excise
Materials and methods Plasmid construction RNA was isolated from cultured skin fibroblast cells originating from a healthy female volunteer with the RNeasy Total RNA Kit (Qiagen, Hilden, Germany). XPF cDNA was synthesized with an oligo(dT) primer using AMV reverse transcnptase, followed by amplification of the cDNA with two primers, 5'-ATAGGTACCGGCTCGACGGATTGCCAT-3' and 5'-CGCTCTAGATGTCTGGCAAGGAGCCGCT-3', by the long and accurate polymerase chain reaction (LA-PCR) protocol (Takara, Kyoto, Japan). As the amplified XPF cDNA (2.8 kb) has a Kpn\ restriction site at the 5'-end and an Xba\ restriction site at the 3'-end, the cDNA was digested with these restriction endonucleases and ligated into an expression vector plasmid pcDNA3 (Invitrogen, San Diego, CA) that had been digested with the same endonucleases. The plasmid was designated pcDNA3-XPF and XPF cDNA is transcribed by a cytomegalovirus promoter when transfected into human cells. Nucleotide sequences of the XPF cDNA were determined with the DyeTerminator Cycle Sequencing FS Kit (Applied Biosystems) with a 373A automated DNA sequencer (Applied Biosystems). In the XPF cDNA, polymorphisms were found at codons 24 (TGT), 321 (AGT), 692 (GGC) and 830 (TCT). The base change at codon 692 was also reported by Sijbers et al. (1996a). Other changes do not cause amino acid changes. Plasmid pBluescript-ERCCl was supplied from Dr R.D.Wood. The ERCC I cDNA (1.0 kb) was cut out from the plasmids with restriction endonucleases BstXl and Xhol and ligated to the expression vector plasmid pcDNA3 (Invitrogen, San Diego, CA) that had been digested with the same endonucleases. The plasmid was designated pcDNA3-ERCC 1. Plasmid transfection The simian virus 40 (SV40)-transformed XP-F fibroblast cell line XP2YCHSV) (Yagi and Takebe, 1983), v.hich had been stored in our laboratory since their establishment, were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. The cells were trypsinized, washed and suspended in phosphate-buffered saline (PBS). The cells (2X10'') plus 2 |ig either pcDNA3-XPF or pcDNA3-ERCCl in 0.2 ml PBS were placed in an electroporation chamber (electrodes 0.3 cm apart) and the cells were transfected with the plasmid by four 500 V electric pulses (ZA1200; Toyobo, Osaka, Japan) Oalsuka tt al, 198S). Th=:% the cells wen seeded at 5X105 cells/ 10 cm dish and cultured for 2 weeks in medium containing 400 |ig/ml
T o whom correspondence should be addressed. Tel: ^-81 75 753 4412; Fax: -^-81 75 753 4419; Email: c51845SsaItura.laidpc.kyoto-u.ac.jp © UK Environmental Mutagen Society/Oxford University Press 1998
595
Downloaded from https://academic.oup.com/mutage/article-abstract/13/6/595/1044087 by guest on 23 October 2018
The XPF and ERCC1 proteins form a tight complex and function as an endonuclease to incise on the 5'-side of pyrimidine dimers in DNA. Levels of both proteins are extremely low in group F xeroderma pigmentosum (XP-F) cells. We transfected XP-F cells with the plasmids expressing XPF or ERCC1 and examined levels of both proteins in the cells. Although XP-F cells are sensitive to UV and mitomycin C (MMQ, cells overexpressing XPF expressed ERCC1 as well and resistance to UV and MMC was restored to the normal level. In contrast, cells overexpressing ERCC1 did not express XPF and were still sensitive to UV and MMC. These results indicate that both the XPF and ERCC1 proteins are required to repair UVand MMC-induced DNA damage. Even though a high level of ERCC1, which has been presumed to be a catalytic subunit of the endonuclease, is stably present in XP-F cells, ERCC1 protein alone cannot carry out excision repair completely.
23-32 nt length single-strand DNA fragments (Moggs et aL, 1996; Bessho et al, 1997; Evans et al, 1997). ERCC1 has been presumed to be a catalytic subunit of the endonuclease complex because it has a region homologous to bacterial UvrC protein and the exonuclease domain of DNA polymerases (Sijbers et al., 1996b). The level of ERCC1 protein as well as XPF protein is markedly low in XP-F cells, although transcription of ERCC1 mRNA is normal in XP-F cells (van Duin et al, 1986). ERCC1 is thought to be unstable and is stabilized by binding to XPF protein in normal cells (Biggerstaff et al, 1993; van Vuuren et al, 1993; Yagi et al, 1997). The roles of XPF have not yet been fully clarified. In this study we transfected XP-F cells with plasmids which can express XPF or ERCC1 driven by a strong promoter and the resulting cell clones were used to examine the relationship between the cellular level of the proteins and restoration of DNA repair activity. We have recently reported one of these XPF-overexpressing clones, which showed complete restoration of DNA repair activity (Yagi et al, 1998).
Takashi Yagi el at geneticin Several geneticin-resislant colonies were randomly isolated and cultured for further experiments. The geneticin-resistant cell clones transfecled with pcDNA3-XPF and pcDNA3-ERCCl were designated 2YO/XPFR and 2Y0/ERC1 respectively
Northern blots RNA was isolated from clones 2YO/XPFR and 2YO/ERC1 and XP2YCXSV) (XP-F) and WI38VA13 (normal) cells with the RNeasy Total RNA Kit (Qiagen, Hilden, Germany). The RNA (20 |ig) was separated by 1 % agaroseformaldehyde gel electrophoresis and the RNA was transferred to Biodyne B membrane (Pall, Glen Cove, NY). The RNA on the membrane was hybridized with XPF or ERCCl cDNA labelled with [a-32P]dCTP (3000 Ci/mmol; Amersham, Little Chalfont, UK) with the Redipnme Labelling System (Amersham) in Rapid Hybridization Buffer (Amersham). After washing the membrane, XPF and ERCCl mRNAs were detected by autoradiography. The same membrane was rehybndized with 32P-labelled (J-actin cDNA as a control Immunoblol of XPF and ERCCl proteins Cell extracts were obtained from clones 2YO/XPFR and 2YO/ERC1 and XP2YO(SV) (XP-F) and WI38VA13 (normal) cells The extracts (40 ug) were separated by SDS-PAGE and transferred to Hybond-C Super membrane (Amersham) as described previously (Yagi el al., 1997). The XPF and ERCCl proteins were detected with affinity-purified anti-XPFand anti-ERCCI polyclonal antibodies respectively (kindly supplied by A.N Sijbers) and with a second antibody, a horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin (Santa Cruz Biotechnology, Santa Cruz, CA), and detected with a Renaissance Western Blot Chemiluminescence Kit (Dupont. Boston, MA) PCNA was detected on the same membrane with the anti-PCNA monoclonal antibody PC-10 as a control.
Results Levels of XPF and ERCCl expression in the cell clones obtained by pcDNA3-XPF transfection By transfecting XP2YO(SV) cells with pcDNA3-XPF, seven geneticin-resistant cell clones were isolated, designated 2YO/ XPFR-1-7. Expression of XPF and ERCCl were investigated in six clones, except for clone 2YO/XPFR-6, which did not grow well. Levels of XPF protein and mRNA were examined by western (Figure 1A, top) and northern blotting (Figure IB, top) respectively. VA13 (normal) cells expressed two kinds of XPF mRNA (-7.4 and -3.8 kb), while XP2YO(SV) cells and the clones 2YO/XPFR-4 and 7 expressed one short mRNA (-2 kb). Clones 2YO/XPFR-1, 2. 3 and 5 expressed abundant XPF mRNA (-2.8 kb), which was transcribed from the transfected XPF cDNA in addition to the endogenous 2 kb mRNA. XPF mRNA expression in clones 2YO/XPFR-2 and 3 was extremely high. Corresponding to the expression level of mRNA, expression of the XPF protein (-120 kDa) was detected in clones 2YO/ XPFR-1, 2, 3 and 5 and in VA13 cells. The level of protein expression was very high in clones XP2YO/XPFR-2 and 3 but very low in clone 2YO/XPFR-1. No XPF protein was detected in clones 2YO/XPFR-4 and 7 and in XP2YO(SV) cells. Levels of ERCC1 protein and mRNA in 2YO/XPFR clones were examined by western (Figure 1A, middle) and northern blotting (Figure IB. middle) respectively. ERCCl 596
Levels of XPF and ERCCl expression in the cell clones obtained by pcDNA3-ERCCl transfection By transfection of XP2YO(SV) cells with pcDNA3-ERCCl, 10 geneticin-resistant cell clones were isolated, designated 2YO/ERC1-1-10. Clone 2YO/ERC1-4, which did not grow well, was not used for the experiments. Levels of ERCCl protein and mRNA were examined by western (Figure 2A, middle) and northern blotting (Figure 2B, middle) respectively. VA13 and XP2YO(SV) cells and all 2YO/ERC1 clones expressed ERCCl mRNA (-1.0 kb) and clones 2YO/ERC1 and 2YO/ERC1-6-9 expressed abundant ERCCl mRNA, which was transcribed from the transfected ERCCl cDNA. Corresponding to the expression level of mRNA, ERCCl protein (-42 kDa) was detected in clones 2YO/ERC1-3 and 2YO/ERC1-6-9 and in VA13 cells. The amount of ERCCl protein was extremely low in clones 2YO/ERC1-1, 2, 5 and 10 and in XP2YO(SV) cells. Levels of XPF protein and mRNA in 2YO/ERC1 clones were examined by western (Figure 2A, top) and northern blotting (Figure 2B, top) respectively. Aberrant XPF mRNA (-2 kb) was expressed in all 2YO/ERC1 clones and in XP2YO(SV) cells at almost the same level. No XPF protein was detected in all 2YO/ERC1 clones and in XP2YO(SV) cells. Sensitivity of the cell clones to UV and MMC Sensitivity of the cell clones 2YO/XPFR and 2YO/ERC1 to UV is shown in Figure 3A and B respectively. Clones 2YO/ XPFR-1, 2, 3 and 5, all of which overexpressed XPF and ERCCl proteins, were more resistant to UV than clones 2YO/ XPFR-4 and 7 and XP2YO(SV) cells. The clone 2YO/XPFR1 was slightly more sensitive to UV than other resistant clones and VA13 cells. All 2YO/ERCI clones were as sensitive as XP2YO(SV) cells to UV, although clones 2YO/ERC1-3 and 6-9 expressed a high amount of ERCCl protein. Sensitivity to MMC of the XPF-expressing cell clones 2YO/ XPFR-1 and 3, the ERCCI-expressing cell clones 2YO/ERC17 and 8 and XP2YO(SV) and VA13 cells are shown in Figure 3C. Clones 2YO/XPFR-1 and 3 and VA13 cells were more resistant to MMC than clones 2YO/ERC-7 and 8 and XP2YO(SV) cells. The UV dose and MMC concentration causing 50% inhibition of colony formation (IC50) and relative levels of XPF and ERCCl proteins in cells are summarized in Table I. Discussion By XPF cDNA transfection into XP-F cells we obtained four cell clones which are as resistant to UV as normal cells. We recently reported that one of the cell clones, 2YO/XPFR-2. has completely normal DNA excision repair ability (Yagi et al., 1998). This restoration of UV resistance and DNA repair ability should be due to correction of the defect in the cells by expression of a single XPF cDNA. In XP-F cells, levels of both XPF and ERCCl proteins are extremely low. Two explanations have been proposed for this (Biggerstaff et al.. 1993; Yagi el al., 1997, 1998). One is that ERCCl is unstable unless it binds to XPF protein and the
Downloaded from https://academic.oup.com/mutage/article-abstract/13/6/595/1044087 by guest on 23 October 2018
UV and mitomycm C sensitivity of cells UV sensitivity of cell clones 2YO/XPFR-I-7 and 2YO/ERC1-I-10 was determined by the post-UV colony formation method as described previously (Yagi et al, 1984). Briefly, these cells were seeded at 100-10,000 cells/6 cm dish and incubated for 16 h. Then, the cells were washed with PBS and exposed to germicidal UV light (254 nm) at a dose rate of 3 4 J/m2/s. The cells were cultured for ~12 days until colonies were formed. For measurement of mitomycin C (MMC) sensitivity, the cells were plated as above and treated with serum-free medium containing MMC at 37°C for 1 h followed by incubation in culture medium until colony formation. The colonies were stained with 5% Giemsa solution. The UV and MMC sensitivity of these cells were compared with that of SV40-transformed normal human cells WI38VA13 (Girardi et al, 1965) and parental XP2YCKSV) cells.
mRNA (-1.0 kb) was expressed in all cells at almost the same level. However, ERCCl protein (-42 kDa) was detected in clones 2YO/XPFR-1, 2, 3 and 5 and in VA13 cells corresponding to the level of XPF protein. A small amount of ERCCl protein was detected in clones 2YO/XPFR-4 and 7 and in XP2YO(SV) cells.
UV and mltomycin C sensitivity, XPF and ERCCl expression
lili
Downloaded from https://academic.oup.com/mutage/article-abstract/13/6/595/1044087 by guest on 23 October 2018
Fig. 1. Expression of XPF and ERCCl protein (A) and mRNA (B) detected by western and northern blotting respectively in the 2YO/XPFR cell clones obtained by transfection with pcDNA3-XPF plasmids. Expression of PCNA protein and |}-actin mRNA were also measured as controls. Mw, position of Kaleidoscope molecular weight standards (BioRad, Hercules, CA). Positions of rRNA are also shown as 28S and 18S.
Fig. 2. Expression of XPF and ERCCl protein (A) and mRNA (B) detected by western and northern blotting respectively in the 2YO/ERC1 cell clones obtained by transfection with pcDNA3-ERCCl plasmids. Expression of PCNA protein and p-actin mRNA were also measured as controls. Mw, position of Kaleidoscope molecular weight standards (BioRad, Hercules, CA). Positions of rRNA are also shown as 28S and 18S.
other is that XPF functions as a transcription factor for the ERCCl gene. In clones 2Y0/XPFR-1, 2, 3 and 5, restoration of ERCCl is not at the mRNA level but at the protein level, indicating that the second explanation should be excluded. The first explanation is more plausible, but not verified completely, because ERCCl alone can exist in clones 2YO/ ERC1-3 and 6-9. At the beginning of this study we postulated that over-
expression of ERCCl may restore the excision repair ability of XP-F cells if XPF protein functions only to stabilize ERCCl protein, which has been presumed to act as an endonuclease (Sijbers et ai, 1996b). Figures 1 and 2 show that our postulation was incorrect and suggest that both the XPF and ERCCl proteins are required to restore excision repair in XP-F cells. The levels of both the XPF and ERCCl proteins might correspond to the level of excision repair activity of the 597
Takashi Yagi et aL
A
B 10" )
V \\
10 '
o>-
3
V \
-
— •
VA13
•
• •
-
• —
VA13
•
XP7YO(SV)
»
7YCXPFR t
XPJYCXSV)
•
7¥O€RC
—-•>
2YO.-ERC
7
—o
2YOERC
3
^—
TYOtnC
-6
— - —
nazpc
7
ZYOtnC
-e
2V0*RC
9
;
io
2
1
A
• 10 3 -
— • ±
JYCWPFR 5
—
7YCVXPFR7
— — •
10'4 2 UV
D O H (J/m2)
ZtOtPC
10
« Dot*
3
-
\
-10 10
0 UV
10 ' •
10 ' -
0
*•
0 0 Mitomycln
(J/m2)
V*
V
\
I
10
VA13
•
*P2YO(EVf
*
7YOtRCi 7
^ - O ^
2YO-tHCH
—o—
?iO XPTP i
ft— ?rO-XPFR 3 2 0
3 0
C Concentration (|JM)
Fig. 3. Sensitivity to UV of cell clones 2YO/XPFR (A) and 2YO/ERC1 (B) and sensitivity to MMC of cell clones 2YO/XPFR-1 and 3 and 2YO/ERC1-7 and 8 (C), measured by colony forming ability. Sensitivity of VA13 (normal) and XP2YCKSV) (XP-F) cells was also measured as controls.
Table I. UV dose and MMC concentration causing 50% inhibition of colony formation (IC50) and relative levels of XPF and ERCC1 proteins in cells Cell
2YO/FR-1 2YO/FR-2 2YO/FR-3 2Y0/FR-* 2YO/FR-5 2YO/FR-7 2YO/ERC1-1 2YO/ERC1-2 2YO/ERC1-3 2YO/ERC1-5 2YO/ERC1-6 2YO/ERCI-7 2YO/ERC1-8 2YO/ERC1-9 2YO/ERC1-10 VAI3 XP2YCKSV)
Overproduction11
ICso UV (J/m2)
MMC (uM)
XPF
ERCC1
4.16 7 69 4 78 0.94 5.49 1.18 1.15 1 38 0.86 1.31 0.92 1.00 0 77 1 38 1.03 6.06 1.01
0.24 ND 0.27 ND ND ND ND ND ND ND ND 0.07 0.09 ND ND 0.44 0.13
+ ++ ++
+ ++ ++
++ -
++ -
-
++
++
++ ++ ++ ++ +
ND. not determined. "-, less than twice, + less than 10 times and + + more than 10 times the protein levels of XP2YO(SV) cells estimated from Figures I and 2
cells, because clone 2YO/XPFR-1, having low levels of the proteins, is slightly more sensitive to UV than other XPFoverexpressing clones. Rodent ERCC-I and 4 mutant cells are hypersensitive to MMC, probably because these cells are deficient in the ability to repair DNA interstrand crosslinks (Stefanini et al., 1987; Sijbers et al., 1996b; Busch et al., 1997). Figure 3C shows that XP-F cells are also hypersensitive to MMC compared with normal cells, although the difference in sensitivity between the cells is not so large as that in UV sensitivity. The XPFoverexpressing clones 2YO/XPFR1 and 3 are as resistant to MMC as VA13 cells, but the ERCCl-overexpressing clones 2YO/ERC1-7 and 8 are as sensitive as XP2YO(SV) cells. This suggests that both XPF and ERCC1 are required to repair DNA interstrand crosslinks in human cells. In XP2YO(SV) cells, one species of truncated XPF mRNA (~2 kb) is expressed and XPF protein is undetectable by western blotting. As mutations were found in the latter-half of 598
XPF mRNA in all XP-F patients examined so far (Sijbers et al., 1996a; Matsumura et al., 1998), we have pointed out the indispensability of the N-terminal half of XPF protein for DNA repair (Matsumura et al., 1998). However, the present study indicates that XP2YO(SV) cells, completely lacking XPF protein, still have a low level of nucleotide excision repair ability. As rodent cells completely lacking ERCC1 show extremely high UV sensitivity, the residual excision repair and intermediate UV sensitivity of XP2YO(SV) cells could be caused by the remaining small amount of ERCC1 unbound to XPF protein. Brookman et al. (1996) reported that XP2YO(SV) cells express a low level of XPF protein and that the UV resistance of XP2YO(SV) cells was only partially restored by XPF cDNA transfection. However, our XP2YO(SV) cells do not express a detectable amount of XPF protein and DNA repair capability of the cells is fully corrected by transfection with a single XPF cDNA (Yagi et al, 1998). This contradiction may be ascribed to different genetic changes occurring during longterm culture of XP2YO(SV) cells in different laboratories after SV40 transformation. Parental XP2YO diploid fibroblast cells express two species of normal size XPF mRNA (3.8 kb) containing a one base deletion at site 1937 (codon 646) and a base change (A-G) at site 1666 (codon 556) (Matsumura et al., 1998). Our XP2YO(SV) cells may have a genomic rearrangement in the XPF gene, resulting in expression of a short mRNA (~2 kb). In conclusion, the present study indicates that both the XPF and ERCC1 proteins are indispensable for resistance of cells to UV and MMC and, hence, suggests that both proteins are required to repair UV- and MMC-induced DNA damage. Even if a large amount of ERCC1, which has been presumed to be an incision subunit of the endonuclease (Sijbers et al., 1996b), is stably expressed in XP-F cells, ERCC1 protein alone cannot carry out excision repair completely. Establishment of XPF knockout cells are necessary to substantiate this conclusion. Ackowledgements We thank Drs A.N.Sijbers and R.D.Wood for supplying an anti-XPF antibody and the plasmid pBluescript-ERCCl respectively This study was supported by Grants-in-Aid from the Ministry of Education, Science. Sports and Culture. Japan.
References Aboussekhra,A., Biggerstaff.M , Shivji.M K K , VilpoJ.A., Monocolhn.V. PodusuVN. Protic.M.. Hubscher.U., EglyJ.-M and Wood.R D (1995)
Downloaded from https://academic.oup.com/mutage/article-abstract/13/6/595/1044087 by guest on 23 October 2018
0
10" •
10 v I
UV and mitomydn C sensitlvitj', XPF and ERCC1 expression human fibroblast cells, and a factor affecting the amount of UV-induced unscheduled DNA synthesis. Mutat. Res., 132, 101-112. Yagi.T., WoodJ*.D. and Takebe,H. (1997) A low content of ERCC1 and a 120 kDa protein is a frequent feature of group F xeroderma pigmentosum fibroblast cells. Mutagenesis, 12, 41-44. Yagi.T., Matsumura,Y., Sato,M., Nishigori.C., Mori.T., Sijbers,A.M. and Takebe.H. (1998) Complete restoration of normal DNA repair characteristics in group F xeroderma pigmentosum cells by over-expression of transfected XPF cDNA. Carcinogenesis, 19, 55-60. Received on February 2. 1998; accepted on April 20, 1998
Downloaded from https://academic.oup.com/mutage/article-abstract/13/6/595/1044087 by guest on 23 October 2018
Mammalian DNA nucleotide excision repair reconstituted with purified protein components. Cell, 80, 859-868. Bardwell.AJ.. Barduell.L., Tomkinson,A.E. and FriedbergJi.C. (1994) Specific cleavage of model recombination and repair intermediates by the yeast Radl-RadlO DNA endonuclease. Science, 265, 2082-2085. Bessho.T, Sancar.A., Thompson.L.H. and Thelen.M.P. (1997) Reconstitution of human excision nucleate with recombinant XPF-ERCC1 complex. J. BioL Chem., Ill, 3833-3837. Biggerstaff.M, Sz>mkov\sLi,D.E. and Wood.R.D. (1993) Co-correction of the ERCC1, ERCC4 and xeroderma pigmentosum group F DNA repair defects in vitm. EMBO J., 12, 3685-3692. Brookman,K.W., LamerdinJ.E., Thelen.M.P., Hwang.M., ReardonJ.T., Sancar^A., Zhou,Z.-Q., Walter.C.A., Parris.C.N. and ThompsonXH. (1996) ERCC4 (XPF) encodes a human nucleotide excision repair protein with eukaryotic recombination homologs. Mol. Cell. BioL, 16, 6553-6562. Busch,D.B., van Vuuren.H., de WitJ., Collins^., ZdzienickaJvI.Z., Mitchell.D.L., Brookman.K.W., Stefanini.M., Riboni,R., ThompsonX-H., Albert,R.B., van Gool.AJ. and HoeijmakersJ. (1997) Phenotypic heterogeneity in nucleotide excision repair mutants of rodent complementation groups 1 and 4. Mutat. Res., 383, 91-106. Davies.A.A., Friedberg.E.C, TomkinsonAE., Wood,R.D. and Stephen.C. (1995) Role of the Radl and RadlO proteins in nucleotide excision repair and recombination. J. Biol. Chem., 270, 24638-24641. Evans.E., FellowsJ., Coffer.A. and Wood.R.D. (1997) Open complex formation around a lesion during nucleotide excision repair provides a structure for cleavage by human XPG protein. EMBO J., 16, 625-638. Girardi.AJ., Jensen.F.C. and KoprowskiJL (1965) SV40-induced transformation of human diploid cells: crisis and recovery. J. Cell. Comp. Physiol., 65, 69-84. HoeijmakersJ.HJ. (1993) Nucleotide excision repair II: from yeast to mammals. Trends Genet., 9, 211-217. Kraemer.K.H., Lee,M.M. and ScottoJ. (1987) Xeroderma pigmentosum. Cutaneous, ocular, and neurologic abnormalities in 830 published cases. Arch. Dermatol., 123, 241-250. Matsumura.Y., Nishigori.C, Yagi.T., Imamura,S. and Takebe.H. (1998) Characterization of molecular defects in xeroderma pigmentosum group F in relation to its clinically mild symptoms. Hum. Mol. Genet., 7, 969— 974, 1998. Matsunaga.T., Park,C.-H., Bessho.T., Mu,D. and Sancar.A. (1996) Replication protein A confers structure-specific endonuclease activities to the XPFERCC1 and XPG subunits of human DNA repair excision nuclease. J. Biol. Chem., 271, 11047-11050. MoggsJ.G., Yarema.KJ., EssigmannJ.M. and Wood.R.D. (1996) Analysis of incision sites produced by human cell extracts and purified proteins during nucleotide excision repair of a 1,3-d(GpTpG)-cisplatin adduct. /. Biol. Chem., 271, 7177-7186. Park,C.-H. and Sancar.A. (1994) Formation of a ternary complex by human XPA, ERCC1 and ERCC4 (XPF) excision repair proteins. Proc. Natl Acad. Sci. USA, 91, 5017-5021. Sijbers.A.M., De Laat,WX., Ariza,R.R., Biggerstaff.M., Wei,Y.-F, MoggsJ.G., Carter.K.C, Shcll.B.K., Evans.E., De Jong.M.C, Rademakers.S., De RooijJ., Jaspers.N.G., HoeijmakersJ.HJ. and Wood.R.D. (1996a) Xeroderma pigmentosum group F caused by a defect in a structure-specific DNA repair endonuclease. Cell, 86, 811-822. SijbersAM., van der SpelcP., Odijk.H., van den BergJ., van Duin.M., WesterveldA-. Jaspers.N.G., Bootsma,D. and HoeijmakersJ.HJ. (1996b) Mutational analysis of the human nucleotide excision repair gene ERCC1. Nucleic Acids Res., 24, 3370-3380. Stefanini.M., Mondello.C, Tessera,M.L., Botta.E. and Nuzzo.F. (1987) Cellular and genetic studies in three UV-sensitive Chinese hamster mutants. Cytotechnology, 1, 91-94. Tatsuka,M., Orita,S., Yagi.T. and KakunagaX (1988) An improved method of electroporation for introducing biologically active foreign genes into cultured mammalian cells. Exp. Cell Res., 178, 154-162. van Duin.M., de WitJ., Odijk.H., Westervcld.A., YasuiA. Koken.M.H.M., Hoeijmaker>J.HJ. and Bootsma,D. M986) Molecular characterization of the human excision repair gene ERCC-1: cDXA cloning and amino acid homology with the yeast DNA repair gene RADIO. Cell, 44, 913-923. van VuurenAJ.. Appeldoorn.E., Odijk,H., YasuiA. Jaspers,N.GJ. and HoeijmakersJ.HJ. (1993) Evidence for a repair enzyme complex involving ERCC1 and complementing activities of ERCC4, ERCC11 and xeroderma pigmentosum group F. EMBO J., 12, 3693-3701. Yaei.T. and Takebe.H. M983l Establishment by SV40-traTT-frrrm2t'nn and characteristics of a cell line of xeroderma pigmentosum belonging to complementation group F. Mutat. Res., 112, 59—66. Yagi.T., Xikaido.O. and Takebe.H. (1984; Excision repair of mouse and
599
