Short communicationXPA protein as a limiting factor for nucleotide excision repair and UV sensitivity in human cells
Introduction
Many agents used for chemotherapy induce DNA damage that is repaired by nucleotide excision repair (NER), one of the major DNA repair pathways in cells. Reducing the efficiency of the NER pathway in tumors might, therefore, increase the efficacy of DNA damaging chemotherapeutic drugs. However, such an approach requires detailed knowledge of the rate-limiting events. NER requires the action of about 30 polypeptides, which function by the stepwise assembly of interacting proteins at a site of DNA damage [1]. It is not known to what extent any of these factors are rate-limiting for the NER reaction in cells.
The 31 kDa XPA protein is part of the core preincision complex of NER and interacts with DNA as well as with other NER protein factors including RPA, TFIIH and ERCC1 [2]. In the absence of XPA, no stable preincision complex can form [3], [4] and no NER occurs. Consequently, cells deficient in XPA protein have no capacity for NER and are hypersensitive to killing by damage caused by UV radiation and chemical mutagens [5]. As a potential target for improving chemotherapy, XPA has some attractions. Unlike most other NER proteins, XPA does not appear to have additional functions in other biochemical processes such as recombination, transcription or DNA replication. Consequently, disruption of XPA function is expected to specifically affect NER, without concomitant effects on other metabolic pathways.
Previous studies suggested that amounts of XPA protein lower than those found in normal cultured cells are sufficient to confer normal resistance to UV radiation and repair of UV-radiation-induced damage [6], [7], [8], [9]. However, a level at which XPA becomes a rate-limiting factor for NER has not been determined. Here, we have used a regulatable system and quantitative immunoblotting to determine the amount of XPA protein that is limiting for repair and sensitivity to UV radiation in living cells.
Section snippets
Cell lines and culture
WI38-VA13, an SV40 transformed normal fibroblast cell line was obtained from ATCC. SV40 transformed fibroblast cells derived from XP-A patient XP12RO (homozygous for a nonsense mutation at the codon for Arg207) and XP129 cells, a revertant cell line derived from XP12RO [10], were obtained from James Cleaver (UCSF). WI38-VA13 was maintained in DMEM medium (Mediatech) supplemented with 10% heat-inactivated fetal calf serum and 1% antibiotics (Penicillin, Streptomycin). XP12RO, XP129 and the
Reducing the amount of XPA protein to 30% of normal does not affect cellular resistance to UV radiation
To obtain cell lines with a regulatable level of XPA protein, the XPA-deficient cell line XP12RO was stably transfected with a tet-regulatable system expressing the XPA cDNA (Fig. 1a). For comparison, endogenous XPA protein was quantified in WI38-VA cells and the XP revertant cell line XP129 (Fig. 1b). The number of XPA molecules per cell in the different cell lines was determined by calibration with purified XPA protein (Fig. 1d, Table 1, and data not shown). Repair-proficient WI38-VA cells
XPA as a rate-limiting factor for NER and UV sensitivity
Under what circumstances is endogenously expressed XPA protein a rate-limiting factor for NER? In human beings, 50% of the normal gene dosage appears adequate as the parents of XP-A individuals are heterozygous for XP gene expression, and do not display acute sensitivity to sunlight or have cutaneous or neurological symptoms [18]. Mice heterozygous for disruption of Xpa do not differ from wild-type littermates in their propensity to develop UV-radiation-induced skin cancer [19], [20].
The issue
Acknowledgments
We thank Stuart Clarkson (Centre Medical Universitaire, Geneva) and Isabelle Dunand-Sauthier for assistance with the DNA repair assays, and our laboratory colleagues for comments on the manuscript.
References (35)
- et al.
Sequential assembly of the nucleotide excision repair factors in vivo
Mol. Cell.
(2001) - et al.
Characterization of reaction intermediates of human excision-repair nuclease
J. Biol. Chem.
(1997) - et al.
Polymorphisms in the human xeroderma pigmentosum group A gene and their impact on cell survival and nucleotide excision repair
DNA Repair (Amst)
(2002) - et al.
Regulated over-expression of DNA polymerase beta mediates early onset cataract in mice
DNA Repair (Amst)
(2003) - et al.
Expression of wild-type p53 is required for efficient global genomic nucleotide excision-repair in UV-irradiated human fibroblasts
J. Biol. Chem.
(1997) - et al.
Single-strand breaks in DNA during repair of UV-induced damage in normal human and xeroderma pigmentosum cells as determined by alkaline DNA unwinding and hydroxylapatite chromatography: effects of hydroxyurea, 5-fluorodeoxyuridine and 1-β-d-arabinofuranosylcytosine on the kinetics of repair
Mutat. Res.
(1979) - et al.
Unique cross-link and monoadduct repair characteristics of a xeroderma pigmentosum revertant cell line
Mutat. Res.
(1987) - et al.
Tumor suppressor p53 dependent recruitment of nucleotide excision repair factors XPC and TFIIH to DNA damage
DNA Repair (Amst)
(2003) - et al.
Xeroderma pigmentosum p48 gene enhances global genomic repair and suppresses UV-induced mutagenesis
Mol. Cell
(2000) - et al.
DDB1–DDB2 (xeroderma pigmentosum Group E) protein complex recognizes a cyclobutane pyrimidine dimer, mismatches, apurinic/apyrimidinic sites, and compound lesions in DNA
J. Biol. Chem.
(2005)
Sequential binding of DNA-repair proteins RPA and ERCC1 to XPA in vitro
Nucleic Acids Res.
Mechanism of open complex and dual incision formation by human nucleotide excision repair factors
EMBO J.
Genomic characterization of the human DNA excision repair-controlling gene XPAC
Gene
Overexpression of the XPA repair gene increases resistance to ultraviolet-radiation in human-cells by selective repair of DNA-damage
Cancer Res.
Mutational analysis of a function of xeroderma pigmentosum group-A (XPA) protein in strand-specific DNA repair
Nucleic Acids Res.
Low amounts of the DNA repair XPA protein are sufficient to recover UV-resistance
Carcinogenesis
A single-site mutation in the XPAC gene alters photoproduct recognition
Mutagenesis
Cited by (78)
DNA repair efficiency associated with reprogrammed osteosarcoma cells
2019, Gene ReportsKinetics and thermodynamics of zinc(II) and arsenic(III) binding to XPA and PARP-1 zinc finger peptides
2016, Journal of Inorganic BiochemistryDNA damage and repair kinetics of the Alternaria mycotoxins alternariol, altertoxin II and stemphyltoxin III in cultured cells
2016, Mutation Research - Genetic Toxicology and Environmental MutagenesisCitation Excerpt :V79 and HepG2 cells were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and cultured in DMEM or DMEM/F12, respectively, containing 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin. SV40 transformed fibroblast cells derived from XP-A patient XP12RO were obtained from James Cleaver (UCSF, San Francisco, USA) [20]. XP12RO cells were transfected with vector pcDNA3.1 for vector control or vector pcDNA3.1(XPA) containing human XPA cDNA to obtain a XPA-proficient subline [21].