INTRODUCTION

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Alkylating agents were among the first compounds found to be efficacious in cancer therapy and remain important components of many modern chemotherapeutic regimens (38). Many members of this class of drugs have bifunctional groups that can react with both strands of the DNA helix and thus form interstrand and intrastrand cross-links. As profound blocks for both transcription and DNA replication, interstrand cross-links (ICLs) appear to represent the primary cytotoxic lesion induced by most bifunctional alkylating agents.

Repair of DNA ICLs has been studied extensively in Escherichia coli (11, 12). Both genetic and biochemical evidence has established a combined nucleotide excision repair (NER)-recombination mechanism for the error-free repair of ICLs, in which the gap, created by the Uvr(A)BC excinuclease, is repaired through recA-mediated recombination with a lesion-free homologous chromosome as the donor (10, 37, 41). Although the NER-recombination pathway appears to be the primary mechanism of cross-link repair in E. coli, recent evidence has suggested a recombination-independent pathway for cross-link repair in which the gap created by the uvr(A)BC excinuclease is repaired by translesion bypass in order to circumvent a deficiency in recombination or a lack of homologous donor sequences (4, 5). In the budding yeast Saccharomyces cerevisiae, members of the RAD52 epistasis group exhibit hypersensitivity to cross-linking agents and ionizing radiation, indicating that repair of ICLs requires homologous recombination. Members of the RAD3 epistasis group, which are deficient in NER, are also highly sensitive to cross-link damage (21, 29, 36). Taken together, these observations support a combined NER-recombination mechanism for ICL repair in lower eukaryotes. As is the case in E. coli, recombination-independent mechanisms may also play a role in cross-link removal in eukaryotes. The yeast pso1 mutant is characterized by hypersensitivity to psoralen-induced ICLs (19), and genetic analysis has demonstrated allelism between the pso1-1 mutant and the rev3-1 mutant (9). The REV3 gene encodes the catalytic subunit of yeast translesion DNA polymerase  whose function is required for induced mutagenesis (30, 33). A more recent study has indicated that rev3p is important for the processing of ICL repair intermediates in nonreplicating cells, further substantiating a lesion bypass-based recombination-independent mechanism of ICL repair in yeast (28). A plausible role for the rev3p-polymerase  in cross-link repair is the translesion synthesis past the lesion upon the uncoupling of a cross-link.

In mammals, mechanisms of ICL repair are largely unknown. The combined NER-recombination model does not appear to be the major mechanism of repair since the majority of NER mutants exhibit only mild sensitivity to cross-linking agents, although mutants defective in either ERCC1 or XPF do exhibit extreme sensitivity (2, 20). However, several lines of evidence have suggested the involvement of homologous recombination in ICL repair (39). In vivo, elevated levels of sister chromosome exchange induced by bifunctional alkylating agents have been well documented and connected to the repair of ICLs (7, 14, 25). Using a triplex-mediated psoralen cross-link as the model damage, intramolecular homologous recombination has been reported to occur through both nonconservative single-strand annealing and conservative reciprocal exchange pathways (15, 16). Recently, more convincing evidence of recombinational repair of ICLs has emerged through the characterization of two hamster mutants, irs1 and irs1SF, both of which exhibit extreme sensitivity to cross-linking agents (17, 24). Both the XRCC2 and the XRCC3 genes, which complement the repair deficiency of irs1 and irs1SF mutants, respectively, exhibit structural similarity to the hRad51 recombinase family (27). Studies by Johnson et al. (23) and Pierce et al. (34) have indicated that both cell lines harbor a defect in homologous recombination marked by a severe reduction in gene conversion activity. Moreover, RAD54-deficient mouse embryonic stem cells are also hypersensitive to mitomycin C as an apparent result of reduced conservative homologous recombination (14). In addition, Li et al. (26) have shown, in vitro, that the presence of an ICL in a plasmid substrate stimulates repair synthesis in mammalian cell extracts and that this stimulated synthesis is also observed in an undamaged plasmid coincubated in the same extract, suggesting that ICLs can induce recombinational repair synthesis. Taken together, these results provide substantial evidence that recombination factors participate in ICL repair and are likely to be involved in a major pathway of ICL removal.

To investigate the mechanisms by which ICLs are repaired in mammalian cells in the absence of homologous donor sequences, we employed a gene reactivation assay in which a single defined psoralen ICL was introduced into a reporter plasmid in order to block transcription of the reporter gene. Consequently, expression of the reporter gene became dependent upon removal of the ICL. We report here that repair of the ICL present in the plasmid substrate was observed in wild-type cells and that mutants defective in NER were deficient in reactivation, while mutants defective in homologous recombination were not. Rescue and sequencing of repaired plasmids indicated a high rate of mutagenesis at the site of the psoralen cross-link. These results indicate that a recombination-independent, but error-prone, pathway of ICL repair exists in mammalian cells.

	MATERIALS AND METHODS

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Cell lines and tissue culture conditions. Cell lines used in this study were obtained from either the American Type Culture Collection (Rockville, Md.) or the Human Genetic Mutant Cell Repository (Camden, N.J.), unless stated otherwise. The CHO AA8 cell line and its derived mutants UV20 (ERCC1), UV24 (XPB), UV41 (XPF), and UV135 (XPG) were grown in Dulbecco modified Eagle medium medium supplemented with 10% fetal calf serum. The E1KO7-5 and E1KO-47 CHO cell lines were maintained under similar conditions. The human cell lines HT-1080 (fibrosarcoma), RKO (colon cancer epithelial), and the Chinese hamster lung fibroblast cell line V79 were cultured in minimal essential medium (MEM) supplemented with 10% serum. Xeroderma pigmentosum (XP) fibroblast cell lines XP2OS (XPA), XP4PA (XPC), XP6BE (XPD), XP30RO (XPV), and XP3BR (XPG) were maintained in MEM supplemented with 15% serum. The XP30RO cell line was a kind gift from James Cleaver (University of California at San Francisco). The irs1 (XRCC2), and irs1SF (XRCC3) mutants were generously provided by Larry Thompson (Lawrence Livermore National Laboratory, Livermore, Calif.) and Nigel Jones (The University of Liverpool, Liverpool, United Kingdom) and maintained in MEM medium supplemented with 10% serum.

Preparation of cross-linked GFP and luciferase substrates. The cross-linked reporter substrates were prepared by ligation of a psoralen cross-linked 22-mer at a defined position in the plasmid (26). For the green fluorescent protein (GFP) substrate, two complementary oligonucleotides with the following sequences were synthesized and kinased: 5'-GCTCTCGTCTGTACACCGAAG-3' and 5'-GCTCTTCGGTGTACAGACGAG-3'. Boldface letters indicate nucleotide residues involved in the cross-link, and a BsrGI site is underlined. Annealing of these oligonucleotides created identical 3-nucleotide 5' cohesive ends at both ends of the duplex. Then, 100 µg of annealed duplex oligonucleotide (oligo) was mixed with 4,5,8-trimethylpsoralen (Trioxalen) at 5 µg/ml in Tris-EDTA (pH 7.5) buffer containing 25 mM NaCl. The mixture was irradiated with 365-nm UV light (10 min at 10 mW/cm²) to effect formation of the interstrand cross-link between the internal thymidines of the BsrGI site. The cross-linked oligonucleotide was then purified from non-cross-linked DNA by denaturing polyacrylamide gel electrophoresis (PAGE). To insert the cross-linked oligonucleotide, pEGFP-N1 (Clontech) was digested by HindIII, and a single dAMP residue was added to both 3' ends by incubation with Klenow enzyme and dATP. This latter step prevents self-ligation of the plasmid by creating ends that are only ligatable to the cross-linked oligo. After ligation, covalently closed cross-linked plasmid was purified by CsCl-ethidium bromide gradient centrifugation. For construction of the control plasmid, a non-cross-linked oligo was cloned into the HindIII site of the pEGFP-N1 plasmid as described for the cross-linked oligo. To prepare the cross-linked luciferase substrate, two complementary oligonucleotide sequences (5'-TAGCTCGTCTGTACACCGAAG-3' and 5'-TAGCTTCGGTGTACAGACGAG-3') were synthesized and cross-linked as described above. pCMV-luc (a kind gift from M. Hedayati and L. Grossman, Department of Biochemistry, School of Public Health, Johns Hopkins University) was digested by NheI, filled-in with a single dCMP residue, and ligated to the cross-linked oligo. Purification was performed as described above. The control luciferase substrate was prepared by insertion of an uncross-linked oligo in the NheI site. The purity of the cross-linked plasmids was determined by release of the cross-link-containing fragment by restriction enzyme digestion and examination of the products by denaturing PAGE (see Fig. 1).

Transfections and reporter reactivation assay. Transient transfections were performed using FuGENE-6 transfection reagent (Roche Molecular Biochemicals) according to the recommendations of the manufacturer. Carrier DNA was used to equalize the total amount of plasmid DNA when necessary. With the GFP as the reporter, 50 ng of either control or cross-linked plasmid DNA was used to transfect 1.5 × 10⁵ cells seeded in 35-mm plates. For the visualization of the cellular green fluorescence, cells were washed twice with phosphate-buffered saline, fixed in 4% paraformaldehyde at 30 h post transfection, and examined by fluorescence microscopy. For the luciferase reactivation assay, 0.1 to 5.0 ng of cross-linked or unmodified control substrate was used for transfections. Cells were harvested 30 h after transfection, and the luciferase activity was determined by using the Luciferase Assay System (Promega) and measured on a Moonlight 3010 luminometer (Pharmingen). The linear range for the luciferase assay, both in terms of the amount of transfected DNA and the amount of protein extract, was established individually for each cell line. Each datum point was the result of three or more independent transfections.

Mutation analysis. A total of 150 ng of cross-linked pEGFP-N1 substrate was transfected into NER-proficient RKO or HT-1080 cells (5 × 10⁵) in a 60-mm dish. At 30 h after transfection, plasmid DNAs were recovered by either Hirt extraction or a modified alkaline lysis procedure (15) and then electroporated into the E. coli repair-recombination-deficient strain AB2480 (uvrA recA mutant) (18). A 255-bp fragment containing the cross-linked region was generated by PCR amplification directly from the resulting colonies. The PCR products were digested with BsrGI; clones resistant to BsrGI digestion were amplified in kanamycin, and isolated plasmid DNAs were sequenced to identify mutations.

	RESULTS

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Repair of psoralen ICLs in repair-proficient cells. To examine specifically the repair of ICLs, we have developed a procedure to prepare plasmid substrates that contain a single defined ICL (26). Using this method, we placed a single psoralen ICL in the pEGFP-N1 vector between the cytomegalovirus promoter and the GFP coding sequence (see Materials and Methods for the position of the ICL insertion). As shown (Fig. 1A), the purified cross-linked duplex oligo and the resulting plasmid are highly pure and were not found to contain detectable amounts of non-cross-linked material by this examination. As a result of the presence of the cross-link, the expression of the GFP becomes dependent upon the removal of this lesion. Upon transfection of the reporter substrate, pEGFP/CLT, into human repair-proficient HT-1080 cells, a significant number of cells were found to exhibit the GFP signal (Fig. 1B). To determine the efficiency of ICL repair, we inserted the unmodified duplex oligo at the same position in the pEGFP-N1 plasmid. The unmodified reporter plasmid and the cross-linked pEGFP/CLT plasmid were identical except that a single defined ICL was present in the latter. When the same amount of both plasmids was used in parallel to transfect HT-1080 cells, the number of enhanced GFP (EGFP)-positive cells from the cross-linked reporter was estimated to be approximately 30% of that with respect to the unmodified reporter (Fig. 1B). Reactivation of the GFP expression cassette reflected the apparent removal and/or uncoupling of the DNA cross-link and suggested that the cross-links were removed in the absence of a homologous donor sequence, since no significant homology to the pEGFP-N1 plasmid would be expected in a mammalian genome to support homologous recombination. Moreover, sequence alignment of the pEGFP-N1 plasmid indicated that there are no direct repeats flanking the cross-linked region to support an intramolecular recombination process (i.e., single-strand annealing) that could lead to the reactivation of the GFP signal.

View larger version (36K): [in this window] [in a new window]   FIG. 1.   Recombination-independent removal of a single defined psoralen ICL in repair-proficient HT-1080 fibroblast cells. (A) Preparation of cross-linked reporter substrates as examined by denaturing PAGE. The left panel shows a purified cross-linked oligo (lane 2) and a non-cross-linked control (lane 3). Lane 1 contains a 40-mer oligo marker. The right panel shows the purity of the cross-linked plasmid substrate. Lane 1, pEGFP-N1 plasmid with an unmodified oligo inserted at the HindIII site; lane 2, pEGFP-N1 with a cross-linked oligo inserted at the HindIII site. Both were digested with BamHI and NheI to release a 54-bp fragment followed by end labeling with T4 kinase and resolution by 15% denaturing PAGE. (B) Parallel plated HT-1080 cells were transfected with equal amounts of unmodified pEGFP-N1 plasmid (HT-1080/EGFP) or cross-linked pEGFP-N1 plasmid (HT-1080/CLT). Images in the left panels show representative fluorescent views, and images in the right panels show the bright-field view. (C) Repair of a single psoralen ICL in a luciferase reporter plasmid in the repair-proficient cell lines HT-1080, RKO, AA8, and V79. The relative efficiencies of ICL repair were calculated as the percentage of luciferase activity from cross-linked reporter to that of the unmodified reporter.

ERCC1 and XPA mutants are defective in the recombination-independent repair of ICLs. We next used the cross-linked EGFP substrate to examine the repair-deficient UV20 hamster mutant (defective in ERCC1), and a human XP group A mutant, XP2OS. We found that the number of cells expressing EGFP was drastically reduced in the ERCC1 mutant, suggesting a requirement for the ERCC1-XPF heterodimeric complex. Reactivation of the EGFP reactivation was also highly deficient in the XPA mutant, indicating that the XPA gene product was also essential for the recombination-independent removal of ICLs (data not shown).

View larger version (53K): [in this window] [in a new window]   FIG. 2.   Involvement of XPA and ERCC1 in recombination-independent repair of ICLs. The repair of cross-linked luciferase reporter is deficient in XPA and ERCC1 mutants but can be restored to normal levels by the reintroduction of wild-type XPA and ERCC1 genes, respectively. C-XPA, XPA mutant complemented with a XPA cDNA expression vector; E1KO7-5, ERCC1-null mutant derived from CHO ATS-49; E1KO-47 (C-ERCC1), E1KO7-5 mutant transformed by stable integration of a wild-type ERCC1 cDNA expression vector.

Involvement of the NER mechanism in the recombination-independent repair of ICLs. The incision stage of the NER pathway requires the following enzymatic activities (3, 13, 42): the DNA damage-binding activities of XPA-RPA and XPC-hRAD23B, the incision activities of the ERCC1-XPF and XPG endonucleases, and the function of the TFIIH complex, including the helicases XPB and XPD. To determine whether the entire NER incision mechanism is required in the recombination-independent repair of the psoralen ICLs, we examined characteristic human and hamster NER mutants with the cross-linked luciferase reporter. Cross-linked substrate was transfected into each mutant cell line, and reactivation of luciferase activity, as a result of ICL removal, was determined at 30 h posttransfection. As shown (Fig. 3A), reactivation of the cross-linked luciferase reporter, compared to isogenic parental cells, was greatly reduced in hamster mutants UV24, UV41, and UV135, indicating that the functions of the XPB, XPF, and XPG gene products are essential in the recombination-independent repair of ICLs. Consistent with this observation, human mutants defective in either XPD or XPG were also found deficient in the repair-mediated reactivation of luciferase. Thus, both the incision activities of NER, residing in ERCC1-XPF and XPG, and the TFIIH complex appear to participate in the recombination-independent repair of ICLs. Interestingly, the XPC mutant, XP4PA, displayed a partial defect compared to the other mammalian mutants defective in NER. This result is consistent with the observation that XPC function is not required for transcription-coupled repair (TCR) of lesions that reside in the template strand of an actively transcribed gene (32, 40). Since the ICL was placed in the actively transcribed region in the luciferase substrate, detection and processing of the ICL could be carried out by TCR in the absence of XPC. However, XPC apparently contributes to the overall reactivation through the global repair mechanism, as evidenced by the partial reactivation of the cross-linked reporter construct.

View larger version (54K): [in this window] [in a new window]   FIG. 3.   NER mutants are defective in repair reactivation of cross-linked luciferase substrate. (A) Repair efficiency of hamster NER mutants compared to parental AA8 cells. (B) Repair efficiency of XP mutants compared to repair-proficient HT-1080 cells.

irs1 and irs1SF mutants are normal in the recombination-independent repair of ICLs. The irs1 and irs1SF mutants, which are extremely sensitive to cross-linking agents (17, 24), are defective in XRCC2 and XRCC3, respectively. These genes exhibit structural similarity to the hRad51 recombinase family (27), and recent studies have indicated that both mutant cell lines harbor a homologous recombination defect marked by greatly reduced gene conversion activity (23, 34), thus implicating conservative homologous recombination as a major mechanism of ICL repair. To determine whether recombination-independent ICL repair involves the function of the XRCC2 and XRCC3 genes, we examined the irs1 and irs1SF mutants in the luciferase reactivation assay. Both mutants exhibited wild-type recombination-independent repair activity comparable to that of the parental hamster cell lines, V79 and AA8 (Fig. 4). These results suggest that the recombination-independent ICL repair mechanism is likely a pathway distinct from the recombination-dependent mechanisms that require XRCC2 and XRCC3 gene function.

View larger version (54K): [in this window] [in a new window]   FIG. 4.   Recombination-independent repair of ICLs does not require the function of the XRCC2 and XRCC3 genes. Reactivation of the cross-linked luciferase reporter is normal in irs1 and irs1SF mutant cells compared to parental V79 and AA8 cells, respectively.

Recombination-independent repair of ICLs is error-prone. The repair of the cross-linked plasmid substrates that we have observed is most likely mediated by a recombination-independent process, since undamaged sequences that contain significant homology to the cross-linked reporter substrate would not to be available in the reactivation assay. Recombination-independent repair of ICLs is potentially mutagenic since both strands of the helix are damaged by the psoralen ICL. To determine if the recombination-independent repair of ICLs gives rise to mutations at the site of the cross-link, we transfected NER-proficient HT-1080 or RKO cells with the cross-linked pEGFP-N1 substrate. The plasmid DNA was recovered from transfected cells at 30 h posttransfection and transformed into the repair-recombination-deficient E. coli uvrA recA mutant strain AB2480 (18) to propagate lesion-free plasmid DNAs. The thymidine residues where the psoralen cross-link was attached are located within a BsrGI recognition sequence. Any mutation at or around the cross-linked thymidines should result in the inactivation of the BsrGI site. We analyzed 212 independent plasmid clones by PCR and found that approximately half, 108, contained the inserted oligo, while the other half did not. BsrGI digestion of the 108 clones indicated that 9 were refractory to cleavage and thus likely contained mutations in the BsrGI site. Sequencing of the nine clones revealed three A-to-G transversions, four A-to-C transversions, and one A-to-T transition, all of which occurred at the adenine residue opposing a cross-linked thymidine residue (Fig. 5). Interestingly, a strand bias was observed in that, of the eight mutations that occurred opposite a psoralen-adducted thymidine residue, seven were found to be opposite the thymidine residue in the nontranscribed strand, suggesting that the repair of these lesions is largely mediated by TCR as opposed to global repair. The single clone that had a mutation opposite the psoralen-adducted thymidine in the transcribed strand was found to also have a second mutation which was the deletion of a single thymidine residue eight nucleotides from the cross-linked thymidine residue (Fig. 5C). The source of this mutation is not clear, but no other mutations were observed in the sequences of the nine clones that were examined. The ninth mutation observed was a C-to-A transversion which occurred at the cytosine residue immediately 3' to the psoralen-adducted thymidine within the BsrGI site (Fig. 5D). We did not observe deletions involving more than 1 bp as has been reported previously (8, 15), a finding which may be attributed to the lack of plasmid replication in HT-1080 cells, the lack of direct repeat sequences flanking the cross-linked site, and/or our sample size.

View larger version (34K): [in this window] [in a new window]   FIG. 5.   Mutations derived from recombination-independent repair of psoralen ICLs. The rectangle indicates the position of the BsrGI restriction site, and the cross-linked oligonucleotide sequence is underlined. (A) Sequence of the unmodified oligo inserted in the pEGFP-N1 vector. (B) A-to-G transversion found at the site opposite the psoralen-adducted thymidine residue in the nontranscribed strand (triangle). (C) T-to-G transversion found at the site opposite the psoralen-adducted thymidine in the transcribed strand (large triangle), and deletion of a T residue (small triangle). Note that the cross-linked oligo (underlined) was inserted in the opposite polarity. (D) C-to-A transversion found immediately downstream of the A residue opposite the psoralen-adducted thymidine residue (triangle).

	DISCUSSION

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By introducing cross-linked plasmid substrates into cultured cells, we have shown that a single defined ICL was removed through a recombination-independent pathway. Factors that are required for the incision steps of the NER pathway, but not those required for homologous recombination, appear to be essential for this mechanism. In addition, this pathway was shown to be mutagenic since frequent base substitutions were observed at or near the site of the cross-linked residues.

Mechanistic basis of recombination-independent ICL repair. Damage to the DNA duplex can be grouped into two general categories from a molecular perspective. The first category includes damage that affects only one strand of the double helix, such as thymidine dimers, oxidative damage, adducts derived from monofunctional alkylating agents, etc. Repair mechanisms for these lesions rely on the inherent redundancy of the duplex to remove the damaged nucleotide and restore the double helix utilizing the undamaged complementary strand as a template. For instance, nucleotide excision repair, base excision repair, and mismatch repair are able to utilize this mechanism of action. The second category consists of two types of lesions that affect both strands of the double helix: DNA double-strand breaks (DSBs) and ICLs. For the repair of DSBs in mammalian cells, both nonhomologous and homologous recombination pathways have been established as major mechanisms of repair (22). However, the work described here and that reported by others (5, 28) indicates that the repair of ICLs can also be accomplished by recombination-independent mechanisms.

Role of recombination-independent ICL repair in mammalian cells. Repair of ICLs appears to involve two types of mechanisms: error-free repair pathways that involve conservative homologous recombination with the undamaged sister chromatid or homologous chromosome and error-prone pathways that can be either homology dependent or independent, but at the cost of increased genomic instability. Cellular resistance to ICLs is presumably established with a balance between these two types of mechanisms. In mammalian cells, the recombination-dependent mechanism appears to be the predominant pathway for ICL repair. Evidence supporting this notion comes from recent studies of the irs1 and irs1SF mutants, in which the severe cross-link sensitivity of both mutants has been linked to deficiencies in the conservative gene conversion process (23, 34). In contrast, recombination-independent ICL repair seems unlikely as a major pathway for ICL removal because the majority of mammalian NER mutants do not display a severe sensitivity to cross-link damage (2, 20). A plausible explanation is that the NER-mediated ICL uncoupling may be an inefficient process. As indicated above, introduction of the dual incisions by the NER excinucleases requires strand separation at the site of the lesion, and an ICL that covalently joins the two strands together may substantially reduce the introduction of such incisions and/or result in aberrant incisions (6). This, in turn, may lead to a low successful rate of ICL repair through the NER-lesion bypass mechanism. It is also possible that the recombination-independent pathway may function at a specific stage of the cell cycle, perhaps in G₀/G₁, where the homologous recombination repair of ICLs may not be efficient. In budding yeast, the rev3 mutant was found to be more sensitive to the killing by nitrogen mustard in stationary-phase cells than in exponential-phase cells (28), suggesting that recombination-independent ICL repair is employed in a cell cycle phase-specific manner. Moreover, in mutants that are defective in homologous recombination, the rev3p-mediated recombination-independent ICL repair was insufficient to support a normal level of resistance to cross-link damage (28). These results suggest that the recombination-independent ICL appears to be a minor pathway in yeast and that similar mechanisms may also play a minor role in ICL repair in mammalian cells.