Defining the function of XPC protein in psoralen and cisplatin-mediated DNA repair and mutagenesis

Zhiwen Chen, Xiaoxin Susan Xu, Jin Yang and Gan Wang1

Institute of Environmental Health Sciences, Wayne State University, 2727 Second Avenue, Detroit, MI 48201, USA

1 To whom correspondence should be addressed Email: g.wang{at}wayne.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
DNA damage recognition plays an important role in DNA repair and mutagenesis. Failure to recognize DNA damage may lead to DNA replication without damage repair as well as mutation accumulation. Mutations can lead to many disease conditions. XPC is a DNA damage recognition protein that binds to damaged DNA templates at a very early stage during the DNA repair process. We have studied the role of the XPC protein in DNA cross-link reagents, psoralen and cisplatin, mediated DNA repair and mutagenesis. When psoralen and cisplatin-damaged plasmid DNA was transfected into xeroderma pigmentosum group C (XPC) cells, which were defective in the XPC gene, very distinct mutation frequency and spectrum was observed: a decreased mutation frequency for psoralen-damaged plasmid and an increased mutation frequency for cisplatin-damaged plasmid; in contrast, most mutations generated by psoralen in XPC cells were T-to-G transversions and most mutations generated by cisplatin in XPC cells were large deletions. We also determined the DNA repair ability of XPC cells by both host cell reactivation (HCR) assay and in vitro DNA repair assay. The HCR results showed greatly reduced host cell reactivation of a luciferase reporter for both psoralen and cisplatin-damaged plasmid DNA in XPC cells. The in vitro DNA repair results revealed a defective repair capacity for both psoralen and cisplatin-damaged plasmid DNA in nuclear extract prepared from XPC cells. However, this defective DNA repair activity was partially restored when a functional XPC protein was supplemented into the XPC nuclear extract prior to the reaction. These results suggest that the XPC protein DNA damage recognition function plays a crucial role in DNA repair initiation and mutation avoidance and XPC defects may lead to increased mutations and high risk for disease progression.

Abbreviations: EB, ethidium bromide; HCR, host cell reactivation; NF, human normal fibroblast; TFO, triplex-forming oligonucleotide


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In living cells genomic DNA is constantly damaged by both endogenous and exogenous factors. DNA repair plays a very important role in repairing DNA damage and maintaining genetic fidelity and stability (1). One of the critical steps in the DNA repair process is DNA damage recognition (24). The signal of DNA damage recognition will cause cell cycle arrest, allowing DNA repair enzymes to be recruited and the damage to be repaired. If DNA damage is too severe to be repaired, the signal of DNA damage recognition will activate a programmed cell death pathway that will eventually lead to apoptotic cell death. However, if DNA damage cannot be recognized because of defects in DNA damage recognition, cells will escape from cell cycle checkpoint and enter cell cycle without repairing the DNA damage, and in this case, mutations may occur (2). Mutations can lead to many disease conditions (5,6).

In human cells, several DNA damage recognition proteins have been identified. XPA protein, a component of DNA nucleotide excision repair (NER), was identified to have a DNA damage recognition function that initiates the NER process (714). MSH2 protein was also identified to possess a DNA damage recognition function. The MSH2 protein is involved in the DNA mismatch repair process (1519). Several other proteins, including RPA and XPE, were also identified to possess DNA damage recognition function and play important roles in various DNA repair processes (2029).

XPC protein was first identified in xeroderma pigmentosum group C (XPC) cells. The XPC protein binds tightly with HR23B protein to form a stable XPC–HR23B complex (3032). Early studies indicated that the XPC protein functioned as a DNA damage recognition protein for the global genome DNA repair (33,34). However, recent studies suggest that the XPC protein might play a much broader range of role in DNA damage recognition and DNA repair: both in vitro and in vivo DNA repair studies demonstrate that the XPC–HR23B complex is the first component that recognizes and binds to the DNA damage sites during the DNA repair process (3537); biochemical studies indicate an extremely high binding affinity of the XPC protein to damaged DNA templates (2,38,39). In addition, it has been shown that the XPC protein interacts with TFIIH basal transcription factor (4042) and the centrisome protein Centrin 2 (CEN2) (43) inside cells although the exact roles of these interactions in DNA repair has not been determined. In our previous studies, the XPC protein was demonstrated for its involvement in the DNA triplex-mediated transcription-coupled repair process (44). Therefore, it is possible that the XPC protein may function as a universal DNA damage recognition protein and XPC defects may contribute to genetic instability and lead to development of many disease conditions.

In this study, we investigated the role of the XPC protein in DNA cross-link reagents psoralen and cisplatin-mediated DNA repair and mutagenesis using both XPC cells and human normal fibroblast (NF) cells. The mutagenesis study showed very distinct mutation frequencies and mutation spectra for psoralen and cisplatin-mediated mutagenesis in NF and XPC cells. When psoralen-damaged plasmid DNA was transfected into XPC cells, a much lower mutation frequency was observed (0.3% for XPC cells versus 8% for NF cells). In contrast, when cisplatin-damaged plasmid DNA was transfected into XPC cells, a much higher mutation frequency was observed (60% for XPC cells versus 1% for NF cells). The mutation spectra generated by psoralen and cisplatin were also very different in NF and XPC cells: most of the mutations generated by psoralen in NF cells were T-to-A transversions (65%) whereas most of the mutations generated by psoralen in XPC cells were T-to-G transversions (62%); and most of the mutations generated by cisplatin in NF cells were G-to-T transversions (60%) whereas most of the mutations generated by cisplatin in XPC cells were large deletions (<98%). The ability to repair psoralen- and cisplatin-caused DNA damage was also studied in XPC cells using both host cell reactivation (HCR) assay and in vitro DNA repair assay. The HCR results showed a large decrease in luciferase activity for both psoralen and cisplatin-damaged plasmid DNA in XPC cells (5 versus 70% luciferase activity for psoralen-damaged plasmid and 15 versus 66% luciferase activity for cisplatin-damaged plasmid DNA in XPC and NF cells, respectively), indicating decreased DNA repair capability in XPC cells. The in vitro DNA repair assay also demonstrated defective DNA repair activity for both psoralen- and cisplatin-caused DNA damage in XPC nuclear extract. However, this defect was partially restored when purified functional XPC protein was added into the XPC nuclear extract prior to the reactions. All these results suggest that the DNA damage recognition function of XPC protein is crucial for DNA repair and defects of XPC protein lead to a reduced DNA repair capability and increased mutation accumulation. This knowledge may also have important clinical relevance since decreased levels of XPC protein may contribute to the development of many human diseases.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell lines, oligonucleotides and plasmid
The SV40-transformed human normal fibroblast (NF) cells (GM00637) and the primary human XPC cells (GM00671 and GM03176) were obtained from NIGMS Human Genetic Cell Repository (Camden, NJ). The NF cells were maintained in MEM medium supplemented with 10% fetal bovine serum and 2x essential amino acids, non-essential amino acids and vitamins. The XPC cells were maintained in MEM medium supplemented with 20% fetal bovine serum and 2x essential amino acids, non-essential amino acids and vitamins.

The psoralen-conjugated 10mer triplex-forming oligonucleotide, psoAG10, was described previously (45,46) and synthesized by Oligos etc. (Wilsonville, OR). Psoralen was linked with the oligonucleotide at the 5' end via a two-carbon linker arm.

The plasmid pSupFG1 was described previously (45,46). The pSupFG1 plasmid contains a supF reporter gene, an ampicillin resistance gene, a pBR327 replication origin for replication in Escherichia coli and a SV40 viral replication origin/large T-antigen for replication in human cells (Figure 1). The pUSAG15 plasmid was described previously (44). The pUSAG15 plasmid carries a CMV promoter-driven luciferase reporter gene, a psoAG10 binding sequence in front of the luciferase coding region and a pBR327 replication origin for replicating in E.coli (44). The pUSAG15 lacks a mammalian DNA replication origin and, therefore, cannot be replicated in mammalian cells.



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Fig. 1. Structure of pSupFG1 and pUSAG15 plasmids. TBS, the psoAG10 binding site; ori, pBR327 replication origin.

 
Psoralen- and cisplatin-mediated targeted mutagenesis
For psoralen-mediated targeted mutagenesis, the pSupFG1 plasmid DNA was incubated with psoAG10 oligonucleotide (10 µM) in triplex binding buffer (20 mM MgCl2, 10 mM Tris, pH 8.0, 1 mM spermidine) at 37°C for 2 h. The sample was irradiated with long wavelength UV light (UVA, 365 nm, 1.8 J/cm2) (Photochemical reactor RPR-200, Southern New England Ultraviolet Company, Branford, CT) to induce psoralen photoreaction. The unbound psoAG10 was removed from the plasmid by ethanol precipitation. The psoralen-damaged pSupFG1 plasmid DNA was transfected into both NF and XPC cells (20 µg/dish) using the Superfect transfection reagent (50 µl/100 mm dish; Qiagen, Santa Clarita, CA). The transfected cells were incubated at 37°C for 2 days for DNA repair and replication. The plasmid DNA was isolated from the transfected cells using a described protocol (46).

For cisplatin-mediated targeted mutagenesis, the pSupFG1 plasmid DNA was incubated with either 10 or 100 µM of cisplatin (Sigma, St. Louis, MO) at 37°C for 4 h. Free cisplatin was removed from the plasmid DNA by ethanol precipitation. The cisplatin-damaged plasmid DNA was transfected into both NF and XPC cells (20 µg/100 mm dish) using a Superfect transfection reagent (50 µl/100 mm dish; Qiagen). The cells were incubated in a tissue culture incubator (5% CO2 for 2 days at 37°C for DNA repair and replication. Plasmid DNA was isolated from the cells using a described protocol (46).

Detection of mutations occurred in the supF reporter gene
The pSupFG1 plasmid DNA isolated from transfected cells was digested with DpnI restriction enzyme to eliminate any unreplicated plasmid templates. The plasmid DNA was then transformed into an E.coli SY204 (lacZ amber) strain by electroporation with a setting of 1800 V/20 µF and incubated in 1 ml of SOB medium at 37°C for 1 h. The transformed culture was plated on LB agar plates containing both ampicillin (100 µg/ml) and X-gal (20 µg/ml) and incubated at 37°C overnight. As the E.coli SY204 strain carries an amber mutation in the lacZ gene, a functional supF gene suppresses the amber mutation in the lacZ gene and results in blue colonies on the X-gal plate while mutations in the supF reporter gene lead to colorless colonies on the X-gal plates. The mutation frequency of the supF reporter gene was determined as the number of mutant colonies to the number of total colonies on the plates. Plasmid DNA was isolated from the mutant colonies using a QIAprep Spin Miniprep Kit (Qiagen) and analyzed by agarose gel electrophoresis. The plasmids without size change were further sequenced using a dsDNA Cycle Sequencing System (Life Technologies, Gaithersburg, MD) (46).

Dot-blot hybridization assay
The dot-blot hybridization assay was performed under the following conditions. Both undamaged and psoralen- or cisplatin-damaged pSupFG1 plasmid DNA were transfected into NF and XPC cells (10 µg plasmid DNA/100 mm dish) by Superfect reagent (50 µl/dish) for 16 h. The transfection medium was then replaced with fresh cell growth medium. The cells were harvested at various time points after the transfection (24 and 48 h) and plasmid DNA was isolated from the cells. The plasmid DNA was denatured by being resuspended into 500 µl of DNA denaturing solution (0.4 M NaOH, 10 mM EDTA) and boiled in water for 10 min. The denatured DNA samples were loaded to a zeta-probe membrane using a 96-well dot-blot apparatus (Bio-Rad, Hercules, CA). The liquid was filtered through the membrane and removed from the apparatus by vacuum. All wells were rinsed with 500 µl of 0.4 M NaOH solution and the liquid was removed by vacuum until all the wells were mostly dry. The membrane was air-dried for 5 min and the DNA was cross-linked to the membrane by a UV cross-linker (Stratagene, La Jolla, CA). The membrane was pre-hybridized with the hybridization solution [50% formamide, 0.12 M Na2HPO4, pH 7.2, 0.25 M NaCl, 7% (w/v) SDS, 1 mM EDTA] at 43°C for 5 min and then hybridized with the hybridization solution containing the [{alpha}-32P]dCTP labeled pSupFG1 plasmid DNA probe at 43°C overnight. The membrane was removed from the hybridization tube and washed once with each of the following solutions: 2x SSC/0.1%SDS, 0.5x SSC/0.1% SDS and 0.1x SSC/0.1% SDS, at 43°C for 15 min. The membrane was wrapped with Saran Wrap and exposed with X-ray film for detection of pSupFG1 plasmid DNA from each transfection experiment. Quantification of the hybridized radioactive signal was determined using a Kodak EDAS290 Gel Documentation System.

In vitro DNA repair assay
HeLa nuclear extract was purchased from Life Technologies and used as a positive control for the DNA repair assay. The XPC nuclear extract was prepared using a described protocol (47). The in vitro DNA repair assay was performed as described (48). Briefly, the pUSAG15 plasmid DNA was treated with either cisplatin (10 µM) or psoralen to generate DNA damage into the plasmid. Then the DNA repair assay was performed in 25 µl of volume containing 1x HeLa buffer (20 mM HEPES, pH 7.9, 0.1 M KCl, 0.2 mM EDTA), 1 µg of plasmid DNA, 250 µM each dATP, dGTP and dTTP, 10 µCi [{alpha}-32P]dCTP (ICN Pharmaceuticals, Costa Mesa, CA) and 10 µl of nuclear extract. The reactants were incubated at 30°C for 2 h and then digested with Proteinase K (100 µg/ml) at 56°C for 30 min to cleave the proteins contained in the reactants. The reactants were extracted with phenol–chloroform once and the plasmid DNA was purified from the reactants by the Centricon 30 apparatus (Milllipore, Chatsworth, MA). The plasmid DNA was linearized by XhoI restriction enzyme digestion and analyzed by agarose gel electrophoresis using a 0.8% gel. Visualization of plasmid DNA and the incorporated [{alpha}-32P]dCTP was achieved by ethidium bromide (EB) staining and autoradiography. Quantification of the incorporated [{alpha}-32P]dCTP was achieved by phosphorimaging analysis using a Bio-Rad G250 phosphorimager (Bio-Rad).

HCR assay
The HCR assay was performed to determine the DNA repair ability of individual cell lines. For this study, the pUSAG15 plasmid was treated with either psoAG10 plus UVA irradiation or cisplatin to introduce DNA damage into the plasmid DNA. Both the undamaged and the damaged pUSAG15 plasmid DNAs were then transfected into NF and XPC cells (10 µg/transfection reaction) using Superfect transfection reagent. As an internal control, the pRL-CMV plasmid, which carries a renilla luciferase reporter gene, also was co-transfected into the cells with the pUSAG15 plasmid DNA. Effective repairing of psoralen- or cisplatin-generated DNA damage in pUSAG15 plasmid would allow expression of the luciferase reporter gene. In contrast, if the DNA damage was not repaired because of defects in the DNA repair of the host cells, expression of the luciferase gene from the damaged pUSAG15 plasmid would be inhibited. The cells were harvested 2 days after transfection and both firefly luciferase and renilla luciferase activities were determined from the transfected cells using a Dual Luciferase Activity Detection System (Promega, Madison, WI). The activity of firefly luciferase in each experiment was calculated as relative activity to the renilla luciferase activity to minimize the experimental variations. The ratio of luciferase activities in the same cell line for both undamaged and damaged plasmid was used to determine the DNA repair ability of the host cells.

Alkaline gel electrophoresis
The alkaline gel electrophoresis was performed in the following condition. The pSupFG1 plasmid DNA was treated with either psoralen or cisplatin (10 and 100 µM) to introduce DNA damage into the plasmid DNA. Then both undamaged and damaged pSupFG1 plasmid DNA (1 µg) were digested with the XhoI restriction enzyme at 37°C for 3 h. The XhoI digestion cleaved the pSupFG1 plasmid DNA into a 2.5 and a 2.9 kb fragment and the 2.5 kb fragment contained the triplex-forming oligonucleotide (TFO) psoAG10 binding site sequence. The digested DNA samples were analyzed by alkaline gel electrophoresis (50 mN NaOH, 1 mM EDTA) using a 1% gel. The gel was neutralized by soaking in water for 30 min. Visualization of plasmid DNA was achieved by EB staining and autoradiography using a Kodak EDAS290 Gel Documentation System. Quantification of the uncross-linked and cross-linked DNA bands was achieved using the Kodak Gel Documentation System.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Psoralen-mediated targeted mutagenesis of the supF reporter gene in XPC cells
TFOs conjugated with mutagenic chemicals such as psoralen have been used successfully in introducing site-specific DNA damage and targeted mutagenesis (46,4951). This strategy was also used in this work to study the role of XPC protein in psoralen interstrand cross-link-mediated DNA repair and mutagenesis. The TFO psoAG10 was used in this study to introduce a single psoralen interstrand cross-link into the supF reporter gene of pSupFG1 plasmid. Previous studies indicated that over 90% of plasmid carried a single psoralen interstrand cross-link at the triplex binding site when treated with psoAG10 (10 µM) and irradiated with UVA (46,50). The previous DNA repair studies also demonstrated that the triplex structure formed by the psoAG10 did not affect the psoralen DNA damage-mediated DNA repair and mutagenesis process (45). In addition, the triplex structure formed by psoAG10 did not induce any DNA repair or targeted mutagenesis (46,48).

The pSupFG1 plasmid DNA was incubated with psoAG10 oligonucleotide and irradiated with UVA to introduce psoralen interstrand cross-link DNA damage into the plasmid DNA. The damaged plasmid was then transfected into XPC cells and incubated at 37°C for 2 days allowing DNA repair and mutagenesis. As a positive control, the psoAG10-treated pSupFG1 plasmid DNA was also transfected into NF cells for DNA repair and mutagenesis. The undamaged pSupFG1 plasmid DNA was transfected into both XPC and NF cells for determination of the background mutation frequency of the supF reporter gene. Plasmid DNA was isolated from the transfected cells and transformed into an E.coli SY204 strain for detection of mutations occurring in the supF reporter gene (Table I). The background mutation frequency of the supF reporter gene was 0.02% in both NF and XPC cells. When psoralen-damaged pSupFG1 plasmid DNA was transfected into NF cells, the mutation frequency of the supF reporter gene was 8.08%. In contrast, when psoralen-damaged pSupFG1 plasmid DNA was transfected into XPC cells, the mutation frequency of the supF reporter gene was 0.28% in GM00671 XPC cells and 0.31% in GM03176 XPC cells.


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Table I. TFO psoralen-mediated targeted mutagenesis of the supF gene in both NF and XPC cells

 
The supF gene mutations generated by psoralen in both NF and XPC cells were further sequence analyzed (Figure 2). A total of 31 mutations obtained from NF cells were sequenced and 21 of these mutations were T167->A transversions (65%) at the predicted psoralen intercalation site (Figure 2A). A total of 21 mutations obtained from XPC cells were sequenced and 13 of the mutations were T167->G transversions (62%) at the predicted psoralen intercalation site (Figure 2B).



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Fig. 2. Sequence analysis of mutations generated by psoAG10 in the supF reporter gene. (A) Mutations obtained from NF cells (total of 31 mutations). (B and C) Mutations obtained from GM00671 and GM03176 XPC cells (total of 21 mutations). Point mutations produced by psoAG10 are indicated above each base pair, with the listed base representing a change from the top strand. Deletion mutations are presented below the supF sequence and indicated by the dashed lines. The number in parentheses indicated the total number of mutants with the same mutation (underlined). The numbers below the supF gene sequence indicate the position of the supF gene (started at position 91 and ended at position 175).

 
Cisplatin-mediated mutagenesis of the supF reporter gene in NF and XPC cells
The results obtained from the psoralen mutagenesis experiment revealed a decreased mutation frequency for psoralen-mediated mutagenesis in XPC cells. To determine if XPC defects could lead to decreased mutations for other DNA damaging reagents, cisplatin-mediated mutagenesis of the supF reporter gene was studied in XPC cells. The commonly used anticancer drug cisplatin preferentially reacts with the GpG and ApG sequences in DNA to form both intra- and interstrand cross-links (5254). In contrast, psoralen preferentially reacts with thymines at the ApT and TpA sites to form either monoadducts or interstrand cross-links (55).

The pSupFG1 plasmid DNA was treated with cisplatin at either 10 or 100 µM to generate DNA damage in the plasmid DNA. The damaged plasmid DNA was then transfected into both XPC and NF cells for DNA repair and mutagenesis. The plasmid DNA was isolated from the cells and transformed into an E.coli SY204 strain for the detection of mutations occurring in the supF reporter gene (Table II). When the pSupFG1 plasmid DNA was treated with cisplatin at a lower concentration (10 µM), no significant difference was observed for the mutation frequency of cisplatin-mediated mutagenesis in both NF and XPC cells (0.59% for NF cells, 0.76% for GM00671 XPC cells and 0.62% for GM03176 XPC cells) (Table II). When the pSupFG1 plasmid DNA was treated with cisplatin at a higher concentration (100 µM), however, a greatly increased mutation frequency of the supF reporter gene was observed in XPC cells: the mutation frequency of the supF reporter gene in NF cells was 0.97%; in contrast, the mutation frequency of the supF reporter gene was 68% in GM00671 XPC cells and 62.6% in GM03176 XPC cells (Table II).


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Table II. Cisplatin-mediated targeted mutagenesis of the supF reporter gene in both NF and XPC cells

 
Mutations generated by cisplatin in both NF and XPC cells were further analyzed by agarose gel electrophoresis and DNA sequencing assay (Figure 3). The agarose gel electrophoresis results revealed that most of the mutations obtained from NF cells remained unchanged in their plasmid sizes (Figure 3A); however, most of the mutations obtained from XPC cells contained large deletions in their plasmids (>98%) and only a small proportion of mutations (<2%) contained unchanged plasmids (Figure 3B). The mutations obtained from NF cells were further sequence analyzed (Figure 3C). A total of 15 mutations were sequenced and nine of the mutations carried single base G->T transversions (60%).



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Fig. 3. Determination of mutations generated by cisplatin in the supF reporter gene. Determination of plasmid sizes for mutations generated by cisplatin in both NF (A) and XPC (B) cells by agarose gel electrophoresis. (C) Sequence analysis of mutations generated by cisplatin in the supF reporter gene in NF cells (total of 15 mutations). Point mutations are indicated above each base pair, with the listed base representing a change from the top strand. Deletion mutations are presented below the supF sequence and are indicated by the dashed lines. The numbers below the supF gene sequence indicate the position of the supF gene (started at position 91 and ended at position 175).

 
Determination of the damaged plasmid DNA replication in XPC cells
The results obtained from the mutagenesis experiments revealed very different mutation frequencies of DNA cross-link reagents psoralen and cisplatin in XPC cells. To determine whether the damaged plasmid was effectively replicated in the XPC cells, a dot-blot hybridization assay was performed. Both undamaged and psoralen- or cisplatin-damaged pSupFG1 plasmid DNA (10 µg) was transfected into NF and XPC cells. At different time points after the transfection (24 and 48 h), the plasmid DNA was recovered from the transfected cells and a dot-blot hybridization assay was performed to determine the quantification of pSupFG1 plasmid obtained from the transfected cells. As shown in Figure 4, the psoralen-damaged pSupFG1 plasmid DNA recovered very well in both NF and XPC cells in comparison with the undamaged pSupFG1 plasmid-transfected NF and XPC cells. When psoralen-damaged pSupFG1 plasmid DNA was transfected into the cultured cells for 2 days, for example, the plasmid DNA recovered from NF and XPC cells were 74 and 79% to that of undamaged pSupFG1 plasmid in NF and XPC cells, respectively (Figure 4A and C). In contrast, the cisplatin-damaged pSupFG1 plasmid DNA replicated poorly in both NF and XPC cells. When the cisplatin (100 µM)-treated pSupFG1 plasmid DNA was transfected into both NF and XPC cells for 2 days, for example, only 3.3 and 0.3% of the pSupFG1 plasmid was recovered from NF and XPC cells, in comparison with the undamaged pSupFG1 plasmid transfected into NF and XPC cells, respectively (Figure 4A–C).



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Fig. 4. Detection of the plasmid DNA replication for both psoralen and cisplatin-damaged pSupFG1 plasmid in NF and XPC cells. Both undamaged and damaged pSupFG1 plasmid DNA (either psoralen or cisplatin-damaged pSupFG1 plasmid DNA) was transfected into NF and XPC cells. The cells were incubated for various times after the transfection (24 and 48 h) and the plasmid DNA was isolated from the transfected cells. A dot-blot hybridization assay was performed to determine the plasmid DNA replication rate for both psoralen and cisplatin-damaged pSupFG1 plasmid in NF and XPC cells. (A) Autoradiogram of the dot-blot hybridization membrane (1 h exposure time). (B) Autoradiogram of the dot-blot hybridization membrane for the cisplatin-damaged plasmid at longer exposure time (24 h). (C) Relative replication rate of the plasmid DNA in both NF and XPC cells. The amount of the plasmid DNA isolated from the undamaged plasmid-transfected cells was calculated as 100% and the amount of plasmid DNA recovered in the damaged plasmid-transfected cells was calculated as percentage to the amount of the plasmid isolated from the undamaged plasmid-transfected cells. The results are from two individual experiments.

 
Determining the DNA repair ability of XPC cells
To determine whether the observed mutagenesis results were caused by the inability of DNA damage recognition and DNA repair in XPC cells, the DNA repair ability of XPC cells was determined using both HCR assay and in vitro DNA repair assay.

The DNA repair ability of XPC cells was first determined by the HCR experiment. The pUSAG15 plasmid was used in this study because this plasmid carried a CMV-promoter driven luciferase reporter gene and a psoAG10 binding site (44). Both psoralen and cisplatin-damaged pUSAG15 plasmid DNA was transfected into NF and XPC cells for DNA repair and luciferase gene expression. As a control, undamaged pUSAG15 plasmid DNA also was transfected into both NF and XPC cells for luciferase expression. Repairing of psoralen and cisplatin-caused interstrand cross-links in the plasmid would lead to expression of luciferase. In contrast, if the interstrand cross-link DNA damage was not repaired in the plasmid, expression of luciferase would be inhibited. The results of the HCR experiment are shown in Table III. When psoralen-damaged pUSAG15 plasmid DNA was transfected into NF cells, ~87% of luciferase activity was detected in comparison with NF cells transfected with the undamaged pUSAG15 plasmid. When the psoralen-damaged pUSAG15 plasmid DNA was transfected into GM00671 XPC cells, 6% of luciferase activity was detected in comparison with the undamaged plasmid DNA-transfected GM00671 XPC cells. When the psoralen-damaged pUSAG15 plasmid DNA was transfected into GM03176 XPC cells, 16% of luciferase activity was detected in comparison with undamaged plasmid DNA-transfected GM03176 XPC cells. Similar results were obtained when the cisplatin-damaged plasmid DNA was transfected into NF and XPC cells: transfection of cisplatin-damaged pUSAG15 plasmid DNA into NF cells did not cause any reduction of the luciferase activity (100%); in contrast, when the cisplatin-damaged pUSAG15 plasmid DNA was transfected into XPC cells, a much reduced luciferase activity was detected (15%). These results suggested that the DNA repair ability of XPC cells was greatly reduced because of defects in the XPC protein.


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Table III. The efficiency of reactivating luciferase for psoralen and cisplatin-damaged pUSAG15 plasmid DNA in NF and XPC cells determined by host reactivation (HCR) assay

 
The DNA repair ability of XPC cells also was determined using in vitro DNA repair assay. The HeLa nuclear extract was used in this study as a DNA repair proficient control as our previous studies have demonstrated that the psoAG10-generated interstrand cross-link DNA damage was repaired normally in the HeLa nuclear extract (45). The pSupFG1 plasmid DNA was treated with either psoralen or cisplatin (10 µM) to introduce DNA damage into the plasmid template. Then the plasmid DNA was incubated in XPC nuclear extract that was supplemented with [{alpha}-32P]dCTP for DNA repair synthesis (Figure 5). As a positive control, the damaged pSupFG1 plasmid DNA also was incubated in HeLa nuclear extract, which was proficient in DNA repair. Both psoralen and cisplatin-damaged pSupFG1 plasmid DNAs were effectively repaired in HeLa nuclear extract in comparison with the untreated pSupFG1 plasmid (7.6- and 2.7-fold higher than the background) (Figure 5B, lane 2 versus lane 1 and lane 6 versus lane 5). In contrast, very low DNA repair activity was detected when the psoralen or cisplatin-damaged pSupFG1 plasmid DNA was incubated in XPC nuclear extract (2.4- and 0.42-fold of the background) (Figure 5B, lane 3 versus lane 2 and lane 1 and lane 8 versus lane 6 and lane 5). When the purified functional XPC protein was added into the XPC nuclear extracts prior to the reaction, however, the DNA repair defect was partially corrected (4.8- and 1.87-fold higher than the background) for both psoralen and cisplatin-damaged plasmid (Figure 5B, lane 4 versus lane 3 and lane 2 and lane 9 versus lane 8). This result suggested that a functional XPC protein was critical for repairing both psoralen and cisplatin-generated DNA damage in the plasmid and defects of XPC resulted in decreased DNA repair activity.



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Fig. 5. Psoralen and cisplatin-mediated DNA repair of pUSAG15 in nuclear extracts prepared from both HeLa and XPC cells. The pUSAG15 plasmid DNA was treated with 10 µM cisplatin to generate cisplatin DNA damage in the plasmid template. (A) Visualization of the plasmid DNA by EB staining and autoradiography. (B) Autoradiogram of the same gel showing labeled nucleotide incorporation indicative of DNA repair synthesis. (C) Quantification of the incorporated [{alpha}-32P]dCTP in the plasmid DNA. The amount of incorporated [{alpha}-32P]dCTP in pUSAG15 in HeLa nuclear extract was taken as background (100%). Incorporation of [{alpha}-32P]dCTP in other reactions was calculated as percentage to the background. Lane 1, untreated pUSAG15 DNA + HeLa nuclear extract; lane 2, cisplatin (10 µM)-treated pUSAG15 plasmid DNA + HeLa nuclear extract; lane 3, cisplatin (10 µM)-treated pUSAG15 plasmid DNA + XPC nuclear extract; lane 4, cisplatin (10 µM)-treated pUSAG15 plasmid DNA + XPC nuclear extract + purified XPC protein.

 
Characterization of DNA damage generated by psoralen and cisplatin
We also characterized the DNA damage generated by psoralen and cisplatin, especially cisplatin at different concentrations. The pSupFG1 plasmid DNA was treated with either psoAG10 and UVA or cisplatin (10 and 100 µM) to generate DNA damage into the plasmid DNA. Then the plasmid DNA was digested with XhoI restriction enzyme. As a control, the undamaged pSupFG1 plasmid DNA also was digested with the XhoI restriction enzyme. The digested DNA samples were analyzed by alkaline gel electrophoresis using a 1% gel (Figure 6). Digestion of the pSupFG1 plasmid DNA with XhoI generated two DNA fragments, a 2.5 kb fragment and a 2.9 kb fragment (Figure 6A, lane 1). The 2.5 kb DNA fragment contained a psoAG10 binding site sequence, and therefore, could be cross-linked by the psoAG10 and UVA irradiation. Treatment of the pSupFG1 plasmid DNA with psoAG10 and UVA irradiation caused a shift mainly in the 2.5 kb DNA fragment (93%) with a small proportion of 2.9 kb DNA fragment (24%) (Figure 6A and B, lane 4). When the pSupFG1 plasmid DNA was treated with cisplatin at 10 µM, a small proportion of both DNA fragments was shifted from lower bands to higher bands (31%), which indicated the formation of interstrand cross-links (Figure 6A and B, lane 2). When the pSupFG1 plasmid DNA was treated with cisplatin at 100 µM; however, a much higher proportion of both DNA fragments was shifted from the lower bands to higher bands (79%), indicating the formation of an increased amount of interstrand cross-links with the high concentration cisplatin treatment (Figure 6A and B, lane 3). These results suggest that as the concentration of cisplatin increased, the interstrand cross-links generated by cisplatin also increased. The detection of interstrand cross-link DNA damage in both DNA fragments with the cisplatin treatment also suggested a more random distribution of DNA damage generated by cisplatin than the specific DNA damage generated by psoAG10 in the plasmid.



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Fig. 6. Characterization of DNA damage generated by psoAG10 and cisplatin in pSupFG1 plasmid DNA. The pSupFG1 plasmid DNA was damaged by either psoAG10 or cisplatin (10 and 100 µM). Then both undamaged and damaged plasmid DNA was digested with XhoI and analyzed by alkaline gel electrophoresis using a 0.8% gel. (A) Visualization of the plasmid DNA by EB staining and autoradiography. (B) Quantification of the shifted DNA fragments in each damage-treated plasmid. The amount of shifted DNA fragment was calculated as percentage to the total amount of DNA used in the gel. Lane 1, undamaged pSupFG1 plasmid DNA; lane 2, 10 µM cisplatin-treated pSupFG1 plasmid DNA; lane 3, 100 µM cisplatin-treated pSupFG1 plasmid DNA; lane 4, psoAG10-damaged pSupFG1 plasmid DNA. The results are from two individual experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this work, we have investigated the function of XPC protein in DNA cross-link reagents, psoralen and cisplatin, mediated DNA repair and mutagenesis. We first studied psoralen-mediated mutagenesis in XPC cells. The results obtained from our mutagenesis experiments revealed a decreased mutation frequency for psoralen-mediated mutagenesis in XPC cells. This result disagreed with our hypothesis as we speculated that the defect of DNA damage recognition in XPC protein would cause DNA damage recognition failure, leading to DNA replication without the damage being repaired and resulting in increased mutations in XPC cells. To determine if the XPC defect also caused decreased mutation frequency to other DNA cross-link reagent treatments, we further studied cisplatin-mediated mutagenesis in XPC cells. Unlike psoralen, which reacts predominantly with the thymines at the ApT and TpA sites of DNA to form thymine monoadducts and thymine–thymine interstrand cross-links, the anticancer drug cisplatin predominantly reacts with the GpG or ApG sequences to form both intrastrand and interstrand cross-links. When the 10 µM cisplatin-treated pSupFG1 plasmid DNA was transfected into NF and XPC cells for mutagenesis, no significant difference was observed for the mutation frequency in NF and XPC cells (0.59% in NF cells and 0.62–0.76% in XPC cells). When the 100 µM cisplatin-treated pSupFG1 plasmid DNA was transfected into NF and XPC cells for mutagenesis, however, a much increased mutation frequency was observed in the XPC cells: the cisplatin-caused mutation frequency of the supF reporter gene in NF cells was 0.97%; in contrast, the cisplatin-mediated mutation frequency of the supF reporter gene was 68% in GM00671 XPC cells and 62% in GM03176 XPC cells. This result strongly supports our original hypothesis that the XPC defect leads to increased mutations. It also suggested that the decreased mutation frequency for psoralen-mediated mutagenesis in XPC cells might be caused by other mechanisms. One of the possible mechanisms might be the ‘A’ rule in DNA bypass repair process. As psoralen-generated thymine–thymine interstrand cross-links were not recognized in XPC cells due to a defect in the XPC gene, these interstrand cross-links could not be repaired by normal DNA repair processes such as the combination of NER and recombination repair process (56,57). However, these interstrand cross-links still needed to be bypassed for successful DNA replication. The ‘A’ rule might be used in this bypass DNA repair process to incorporate an ‘A’ in the newly synthesized DNA strand to pair with the damaged ‘T’, resulting in undetectable mutations for psoralen-mediated mutagenesis and decreased mutation frequency in XPC cells. In case of cisplatin-mediated mutagenesis, as most of the damage caused by cisplatin is at the GpG or ApG sites, incorporation of ‘A’ to pair with the cisplatin-damaged bases such as G or A still led to detectable mutation, and therefore, resulting in increased mutation frequency in XPC cells. Indeed, most of the single base mutations obtained from our cisplatin mutagenesis experiment in NF cells were G-to-T mutations, indicating the incorporation of ‘A’ to pair with the damaged ‘G’ base and suggesting a possible involvement of the ‘A’ rule in the bypass DNA repair process for cisplatin-generated interstrand cross-links. However, more systematic studies are needed to define the function of the ‘A’ rule in the DNA interstrand cross-link-mediated repair and mutagenesis process.

Mutations generated by psoralen in both NF and XPC cells were sequence analyzed. The majority of mutations generated by psoralen in NF cells were T167->A transversions (65%). This result is consistent with the results obtained in our previous studies (45,48,50) and the data published by others (55). The mutations generated by psoralen in XPC cells were predominantly T167->G transversions (62%). This altered mutation spectrum suggests that a novel mechanism might be involved in the mutagenesis process for psoralen-generated interstrand cross-links in XPC cells. Further studies are needed to determine the mechanism for this altered mutation spectrum in XPC cells.

Mutations generated by cisplatin also were analyzed. DNA sequence analysis data indicated that the majority of mutations obtained from NF cells carried single-based substitutions (13 of 15 mutations analyzed) and a small proportion of the mutations carried deletions that lost the supF reporter gene (two of 15 mutations analyzed). Among the single base substitutions, most mutations were located at the GpG or GpA sites (12 of 13 mutations) with G->T transversions (nine of 13 mutations). This result is in agreement with other published data that cisplatin predominantly reacts with the GpG and ApG sequences and generates G->T transversions (52,53). However, most mutations generated by cisplatin in XPC cells were large deletions that lost the entire supF reporter gene (>98%), and only a small proportion of mutations (<2%) carried single base substitutions. To characterize the DNA damage generated by cisplatin, especially cisplatin at high concentrations, an alkaline gel electrophoresis was performed. When plasmid DNA was pre-treated with cisplatin at a lower concentration (10 µM), a high proportion of the plasmid DNA fragments existed as separated single strand DNAs in the denaturing condition (69%), indicating the lack of interstrand cross-links. When the plasmid DNA was pre-treated with cisplatin at a higher concentration (100 µM); however, a high proportion of the plasmid DNA fragments existed as two-strand formation in the denaturing condition, indicating the presence of interstrand cross-links in the plasmid DNA. Therefore, treatment of plasmid DNA with high concentrations of cisplatin caused a shift of the cisplatin-generated DNA damage to interstrand cross-links. It is also worth mentioning that DNA damages generated by cisplatin were more randomly distributed within the plasmid DNA than the DNA damage generated by psoAG10: treatment of pSupFG1 plasmid DNA with cisplatin caused shifts for both 2.5 and 2.9 kb DNA fragments, whereas treatment of pSupFG1 plasmid DNA with psoAG10 caused a shift predominantly for the 2.5 kb DNA fragment, which contained a psoAG10 binding site. Therefore, the large proportions of deletions detected in XPC cells for cisplatin-mediated mutagenesis might reflect the nature of DNA damage and an altered DNA repair process of the cells.

To confirm whether the altered mutagenesis results obtained from the XPC cells was indeed caused by the XPC defect and its inability to initiate DNA repair, the DNA repair ability of XPC cells was determined by both HCR assay and in vitro DNA repair assay. The results obtained from our HCR assay indicated much more reduced luciferase activities for both psoralen and cisplatin-damaged pUSAG15 plasmid in XPC cells than the damaged plasmid in NF cells. The results obtained from our in vitro DNA repair experiment also demonstrated that the XPC nuclear extract had very low DNA repair activity for repairing both psoralen and cisplatin-caused DNA damage in comparison with the repair activity for the damaged plasmid DNA in HeLa nuclear extract. When purified functional XPC protein was added into the XPC nuclear extract prior to the reaction, however, the DNA repair activity of the XPC nuclear extract for repairing both cisplatin and psoralen-damaged plasmid DNA was partially restored. All these results suggest that a functional XPC protein is crucial for DNA damage recognition and DNA repair, and defects in XPC protein could result in DNA damage recognition failure and decreased DNA repair activity.

The results obtained in this study and the results obtained in our previous study (45) indicated that the psoAG10-generated interstrand cross-links were repaired normally in HeLa nuclear extract. However, it was reported that the interstrand cross-links generated by triplex-conjugated psoralen were not repaired in HeLa nuclear extract (58). The mechanism that led to these different results was unknown. It was possible these opposite results were caused by the TFO used in these studies and the design that psoralen was linked with the TFO. In our studies, the purine-containing TFO, AG10, was designed to bind to the DNA homopurine strand at an anti-parallel orientation to form a triplex formation and psoralen was linked with the TFO at the 5' end; therefore, the triplex structure formed by the AG10 was located at the 5' side of the damaged base (thymine). In contrast, the TFO used by Guieysse et al. (58) was a 15mer pyrimidine-containing TFO that binds parallel to the homopurine strand to form a triplex formation and psoralen also was linked with the TFO at the 5' end; therefore, the triplex structure formed by the TFO was located at the 3' side of the damaged base. It was possible that the triplex structure formed at the 3' side of the homopurine strand blocked the 3' incision of that strand, leading to a failed repair process for the psoralen interstrand cross-links. In fact, the results obtained from our previous studies revealed that the 5' incision also was blocked when longer TFOs, such as psoAG30, were used in the study (45).

The XPC protein was originally defined as a DNA damage recognition protein involved in global genome DNA repair (34). However, recent studies suggested that the XPC protein might also play important roles in other DNA repair processes (44) as well as other cell functions (43,59). The XPC–HR23B complex was the first component that recognized and bound to the DNA damage site and its binding recruited other DNA repair components including the XPA, RPA, TFIIH, XPG and XPF–ERCC1 proteins to the site to remove the DNA damage (36,37,41,60). The transgenic mouse studies with the XPC gene knockout mouse showed predisposition of many types of cancer, including skin cancer, liver cancer, and lung cancer (6163). The results obtained from this study revealed greatly altered mutation frequency and mutation spectrum for DNA damaging treatment in XPC-defective cells. All these results suggest that a functional XPC protein is critical for the DNA repair initiation and mutation avoidance and defects in the XPC protein lead to increased mutations and genetic instability, resulting in a high risk for development of many disease conditions. Therefore, the XPC defects may provide an important biomarker for assessment of many human diseases, especially the cancer and age-related diseases. In addition, the results obtained from this study might also provide important understandings for the molecular mechanism of tumorigenesis and tumor cell resistance to chemotherapy and radiation therapy.


    Acknowledgments
 
We thank J.Harrison, and M.George for their assistance in the experiments. We also thank Drs Dharam P.Chopra and Thomas A.Kocarek for their helpful discussion and critical readings of the manuscript and Ms Elizabeth Sherman for her critical reading of the manuscript. Performance of this work was facilitated by the Cell Culture Facility Core and the Imaging and Cytometry Facility Core of the Environmental Health Sciences Center in Molecular and Cellular Toxicology with Human Applications at Wayne State University (P30 ES06639). This work is supported by the grant R01ES09699 from the National Institute of Environmental Health Sciences, NIH to G.W.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Friedberg,E.C., Walker,G.C. and Siede, W. (1995) DNA Repair and Mutagenesis. ASM Press, Washington, D.C.
  2. Batty,D.P. and Wood,R.D. (2000) Damage recognition in nucleotide excision repair of DNA. Gene, 241, 193–204.[CrossRef][ISI][Medline]
  3. Haase,S.B. and Clarke,D.J. (2001) A festival of cell-cycle controls. Trends Cell Biol., 11, 445–446.[CrossRef][Medline]
  4. Hosfield,D.J., Daniels,D.S., Mol,C.D., Putnam,C.D., Parikh,S.S. and Tainer,J.A. (2001) DNA damage recognition and repair pathway coordination revealed by the structural biochemistry of DNA repair enzymes. Prog. Nucleic Acid Res., 68, 315–347.[ISI][Medline]
  5. Doria,G. and Frasca,D. (2001) Age-related changes of DNA damage recognition and repair capacity in cells of the immune system. Mech. Ageing Dev., 122, 985–998.[CrossRef][ISI][Medline]
  6. Khanna,K.K. (2000) Cancer risk and the ATM gene: a continuing debate. J. Natl Cancer Inst., 92, 795–802.[Abstract/Free Full Text]
  7. Tanaka,K., Miura,N., Satokata,I., Miyamoto,I., Yoshida,M.C., Satoh,Y., Kondo,S., Yasui,A., Okayama,H. and Okada,Y. (1990) Analysis of a human DNA excision repair gene involved in group A xeroderma pigmentosum and containing a zinc-finger domain. Nature, 348, 73–76.[CrossRef][ISI][Medline]
  8. Robins,P., Jones,C.J., Biggerstaff,M., Lindahl,T. and Wood,R.D. (1991) Complementation of DNA repair in xeroderma pigmentosum group A cell extracts by a protein with affinity for damaged DNA. EMBO J., 10, 3913–3921.[Abstract]
  9. Jones,C.J. and Wood,R.D. (1993) Preferential binding of the xeroderma pigmentosum group A complementing protein to damaged DNA. Biochemistry, 32, 12096–12104.[ISI][Medline]
  10. Asahina,H., Kuraoka,I., Shirakawa,M., Morita,E.H., Miura,N., Miyamoto,I., Ohtsuka,E., Okada,Y. and Tanaka,K. (1994) The XPA protein is a zinc metalloprotein with an ability to recognize various kinds of DNA damage. Mutat. Res., 315, 229–237.[ISI][Medline]
  11. Cleaver,J.E. and States,J.C. (1997) The DNA damage-recognition problem in human and other eukaryotic cells: the XPA damage binding protein. Biochem. J., 328, 1–12.[ISI][Medline]
  12. Nocentini,S., Coin,F., Saijo,M., Tanaka,K. and Egly,J.M. (1997) DNA damage recognition by XPA protein promotes efficient recruitment of transcription factor II H. J. Biol. Chem., 272, 22991–22994.[Abstract/Free Full Text]
  13. States,J.C., Duffie,E.R., Myrand,S.P., McDowell,M. and Cleaver,J.E. (1998) Distribution of mutations in the human xeroderma pigmentosum group A gene and their relationships to the functional regions of the DNA damage recognition protein. Hum. Mutat., 12, 103–113.[CrossRef][ISI][Medline]
  14. Hermanson-Miller,I.L. and Turchi,J.J. (2002) Strand-specific binding of RPA and XPA to damaged duplex DNA. Biochemistry, 41, 2402–2408.[CrossRef][ISI][Medline]
  15. Alani,E., Chi,N.W. and Kolodner,R. (1997) The Saccharomyces cerevisiae Msh2 protein specifically binds to duplex oligonucleotides containing mismatched DNA base pairs and insertions. Genes Dev., 9, 234–247.
  16. Gradia,S., Acharya,S. and Fishel,R. (1997) The human mismatch recognition complex hMSH2-hMSH6 functions as a novel molecular switch. Cell, 91, 995–1005.[ISI][Medline]
  17. Kolodner,R.D. and Marsischky,G.T. (1999) Eukaryotic DNA mismatch repair. Curr. Opin. Genet. Dev., 9, 89–96.[CrossRef][ISI][Medline]
  18. Wilson,T., Guerrette,S. and Fishel,R. (1999) Dissociation of mismatch recognition and ATPase activity by hMSH2-hMSH3. J. Biol. Chem., 274, 21659–21664.[Abstract/Free Full Text]
  19. Marra,G. and Schar,P. (1999) Recognition of DNA alterations by the mismatch repair system. Biochem. J., 338, 1–13.[CrossRef][ISI][Medline]
  20. Clugston,C.K., McLaughlin,K., Kenny,M.K. and Brown,R. (1992) Binding of human single-stranded DNA binding protein to DNA damaged by the anticancer drug cis-diamminedichloroplatinum (II). Cancer Res., 52, 6375–6379.[Abstract]
  21. He,Z., Henricksen,L.A., Wold,M.S. and Ingles,C.J. (1995) RPA involvement in the damage-recognition and incision steps of nucleotide excision repair. Nature, 374, 566–569.[CrossRef][ISI][Medline]
  22. Burns,J.L., Guzder,S.N., Sung,P., Prakash,S. and Prakash,L. (1996) An affinity of human replication protein A for ultraviolet-damaged DNA. J. Biol. Chem., 271, 11607–11610.[Abstract/Free Full Text]
  23. Wakasugi,M., Shimizu,M., Morioka,H., Linn,S., Nikaido,O. and Matsunaga,T. (2001) Damaged DNA-binding protein DDB stimulates the excision of cyclobutane pyrimidine dimers in vitro in concert with XPA and replication protein A. J. Biol. Chem., 276, 15434–15440.[Abstract/Free Full Text]
  24. Wakasugi,M., Kawashima,A., Morioka,H., Linn,S., Sancar,A., Mori,T., Nikaido,O. and Matsunaga,T. (2002) DDB accumulates at DNA damage sites immediately after UV irradiation and directly stimulates nucleotide excision repair. J. Biol. Chem., 277, 1637–1640.[Abstract/Free Full Text]
  25. Kataoka,H. and Fujiwara,Y. (1991) UV damage-specific DNA-binding protein in xeroderma pigmentosum complementation group E. Biochem. Biophys. Res. Commun., 175, 1139–1143.[ISI][Medline]
  26. Hwang,B.J. and Chu,G. (1993) Purification and characterization of a human protein that binds to damaged DNA. Biochemistry, 32, 1657–1666.[ISI][Medline]
  27. Nichols,A.F., Itoh,T., Graham,J.A., Liu,W., Yamaizumi,M. and Linn,S. (2000) Human damage-specific DNA-binding protein p48. Characterization of XPE mutations and regulation following UV irradiation. J. Biol. Chem., 275, 21422–21428.[Abstract/Free Full Text]
  28. Hwang,B.J., Liao,J.C. and Chu,G. (1996) Isolation of a cDNA encoding a UV-damaged DNA binding factor defective in xeroderma pigmentosum group E cells. Mutat. Res., 362, 105–117.[ISI][Medline]
  29. Payne,A. and Chu,G. (1994) Xeroderma pigmentosum group E binding factor recognizes a broad spectrum of DNA damage. Mutat. Res., 310, 89–102.[CrossRef][ISI][Medline]
  30. Masutani,C., Sugasawa,K., Yanagisawa,J., Sonoyama,T., Ui,M., Enomoto,T., Takio,K., Tanaka,K., van der Spek,P.J. and Bootsma,D.e.a. (1994) Purification and cloning of a nucleotide excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD23. EMBO J., 13, 1831–1843.[Abstract]
  31. van der Spek,P.J., Eker,A., Rademakers,S., Visser,C., Sugasawa,K., Masutani,C., Hanaoka,F., Bootsma,D. and Hoeijmakers,J.H. (1996) XPC and human homologs of RAD23: intracellular localization and relationship to other nucleotide excision repair complexes. Nucleic Acids Res., 24, 2551–2559.[Abstract/Free Full Text]
  32. Li,L., Lu,X., Peterson,C. and Legerski,R. (1997) XPC interacts with both HHR23B and HHR23A in vivo. Mutat. Res., 383, 197–203.[ISI][Medline]
  33. Mu,D., Hsu,D.S. and Sancar,A. (1996) Reaction mechanism of human DNA repair excision nuclease. J. Biol. Chem., 271, 8285–8294.[Abstract/Free Full Text]
  34. Sugasawa,K., Ng,J.M., Masutani,C., Iwai,S., van der Spek,P.J., Eker,A.P., Hanaoka,F., Bootsma,D. and Hoeijmakers,J.H. (1998) Xeroderma pigmentosum group C protein complex is the initiator of global genome nucleotide excision repair. Mol. Cell, 2, 223–232.[ISI][Medline]
  35. Wood,R.D. (1999) DNA damage recognition during nucleotide excision repair in mammalian cells. Biochimie., 81, 39–44.[CrossRef][ISI][Medline]
  36. Volker,M., Mone,M.J., Karmakar,P., van Hoffen,A., Schul,W., Vermeulen,W., Hoeijmakers,J.H., van Driel,R., van Zeeland,A.A. and Mullenders,L.H. (2001) Sequential assembly of the nucleotide excision repair factors in vivo. Mol. Cell, 8, 213–224.[ISI][Medline]
  37. Sugasawa,K., Okamoto,T., Shimizu,Y., Masutani,C., Iwai,S. and Hanaoka,F. (2001) A multistep damage recognition mechanism for global genomic nucleotide excision repair. Genes Dev., 15, 507–521.[Abstract/Free Full Text]
  38. Wakasugi,M. and Sancar,A. (1999) Order of assembly of human DNA repair excision nuclease. J. Biol. Chem., 274, 18759–18768.[Abstract/Free Full Text]
  39. Hey,T., Lipps,G., Sugasawa,K., Iwai,S., Hanaoka,F. and Krauss,G. (2002) The XPC-HR23B complex displays high affinity and specificity for damaged DNA in a true-equilibrium fluorescence assay. Biochemistry, 41, 6583–6587.[CrossRef][ISI][Medline]
  40. Yokoi,M., Masutani,C., Maekawa,T., Sugasawa,K., Ohkuma,Y. and Hanaoka,F. (2000) The xeroderma pigmentosum group C protein complex XPC-HR23B plays an important role in the recruitment of transcription factor IIH to damaged DNA. J. Biol. Chem., 275, 9870–9875.[Abstract/Free Full Text]
  41. Araujo,S.J., Nigg,E.A. and Wood,R.D. (2001) Strong functional interactions of TFIIH with XPC and XPG in human DNA nucleotide excision repair, without a preassembled repairosome. Mol. Cell. Biol., 21, 2281–2291.[Abstract/Free Full Text]
  42. Winkler,G.S., Sugasawa,K., Eker,A.P., de Laat,W.L. and Hoeijmakers,J.H. (2001) Novel functional interactions between nucleotide excision DNA repair proteins influencing the enzymatic activities of TFIIH, XPG and ERCC1-XPF. Biochemistry, 40, 160–165.[CrossRef][ISI][Medline]
  43. Araki,M., Masutani,C., Takemura,M., Uchida,A., Sugasawa,K., Kondoh,J., Ohkuma,Y. and Hanaoka,F. (2001) Centrosome protein centrin 2/caltractin 1 is part of the xeroderma pigmentosum group C complex that initiates global genome nucleotide excision repair. J. Biol. Chem., 276, 18665–18672.[Abstract/Free Full Text]
  44. Wang,G., Chen,Z., Zhang,S., Wilson,G.L. and Jing,K. (2001) Detection and determination of oligonucleotide triplex formation-mediated transcription-coupled DNA repair in HeLa nuclear extracts. Nucleic Acids Res., 29, 1801–1807.[Abstract/Free Full Text]
  45. Wang,G. and Glazer,P.M. (1995) Altered repair of targeted psoralen photoadducts in the context of an oligonucleotide-mediated triple helix. J. Biol. Chem., 270, 22595–22601.[Abstract/Free Full Text]
  46. Wang,G., Levy,D.D., Seidman,M.M. and Glazer,P.M. (1995) Targeted mutagenesis in mammalian cells mediated by intracellular triple helix formation. Mol. Cell. Biol., 15, 1759–1768.[Abstract]
  47. Dignam,J.D., Lebovitz,R.M. and Roeder,R.G. (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res., 11, 1475–1489.[Abstract]
  48. Wang,G., Seidman,M.M. and Glazer,P.M. (1996) Mutagenesis in mammalian cells induced by triple helix formation and transcription-coupled repair. Science, 271, 802–805.[Abstract]
  49. Havre,P.A. and Glazer,P.M. (1993) Targeted mutagenesis of simian virus 40 DNA mediated by a triple helix-forming oligonucleotide. J. Virol., 67, 7324–7331.[Abstract]
  50. Havre,P.A., Gunther,E.J., Gasparro,F.P. and Glazer,P.M. (1993) Targeted mutagenesis of DNA using triple helix-forming oligonucleotides linked to psoralen. Proc. Natl Acad. Sci. USA, 90, 7879–7883.[Abstract/Free Full Text]
  51. Majumdar,A., Khorlin,A., Dyatkina,N., Lin,F.L., Powell,J., Liu,J. et al. (1998) Targeted gene knockout mediated by triple helix forming oligonucleotides. Nature Genet., 20, 212–214.[CrossRef][ISI][Medline]
  52. Fichtinger-Schepman,A.M., van der Veer,J.L., den Hartog,J.H., Lohman,P.H. and Reedijk,J. (1985) Adducts of the antitumor drug cis-diamminedichloroplatinum (II) with DNA: formation, identification and quantitation. Biochemistry, 24, 707–713.[ISI][Medline]
  53. Chu,G. (1994) Cellular responses to cisplatin. The roles of DNA-binding proteins and DNA repair. J. Biol. Chem., 269, 787–790.[Abstract/Free Full Text]
  54. Kohn,K.W. (1996) Beyond DNA cross-linking: history and prospect of DNA-targeted cancer treatment. Cancer Res., 56, 5533–5546.[Abstract]
  55. Hearst,J.E., Isaacs,S.T., Kanne,D., Rapoport,H. and Straub,K. (1984) The reaction of the psoralens with deoxyribonucleic acid. Q. Rev. Biophys., 17, 1–44.[ISI][Medline]
  56. Wang,X., Peterson,C.A., Zheng,H., Nairn,R.S., Legerski,R.J. and Li,L. (2001) Involvement of nucleotide excision repair in a recombination-independent and error-prone pathway of DNA interstrand cross-link repair. Mol. Cell. Biol., 21, 713–720.[Abstract/Free Full Text]
  57. Kuraoka,I., Kobertz,W.R., Ariza,R.R., Biggerstaff,M., Essigmann,J.M. and Wood,R.D. (2000) Repair of an interstrand DNA cross-link initiated by ERCC1-XPF repair/recombination nuclease. J. Biol. Chem., 275, 26632–26636.[Abstract/Free Full Text]
  58. Guieysse,A.L., Praseuth,D., Giovannangeli,C., Asseline,U. and Helene,C. (2000) Psoralen adducts induced by triplex-forming oligonucleotides are refractory to repair in HeLa cells. J. Mol. Biol., 296, 373–383.[CrossRef][ISI][Medline]
  59. Adimoolam,S. and Ford,J.M. (2002) p53 and DNA damage-inducible expression of the xeroderma pigmentosum group C gene. Proc. Natl Acad. Sci. USA, 99, 12985–12990.[Abstract/Free Full Text]
  60. Wood,R.D., Mitchell,M., Sgouros,J. and Lindahl,T. (2001) Human DNA repair genes. Science, 291, 1284–1289.[Abstract/Free Full Text]
  61. Sands,A.T., Abuin,A., Sanchez,A., Conti,C.J. and Bradley,A. (1995) High susceptibility to ultraviolet-induced carcinogenesis in mice lacking XPC. Nature, 377, 162–165.[CrossRef][ISI][Medline]
  62. Cheo,D.L., Burns,D.K., Meira,L.B., Houle,J.F. and Friedberg,E.C. (1999) Mutational inactivation of the xeroderma pigmentosum group C gene confers predisposition to 2-acetylaminofluorene-induced liver and lung cancer and to spontaneous testicular cancer in Trp53–/– mice. Cancer Res., 59, 771–775.[Abstract/Free Full Text]
  63. Friedberg,E.C., Bond,J.P., Burns,D.K., Cheo,D.L., Greenblatt,M.S., Meira,L.B., Nahari,D. and Reis,A.M. (2000) Defective nucleotide excision repair in xpc mutant mice and its association with cancer predisposition. Mutat. Res., 459, 99–108.[ISI][Medline]
Received May 30, 2002; accepted March 14, 2003.