Human Molecular Genetics Advance Access originally published online on November 25, 2003
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Human Molecular Genetics, 2004, Vol. 13, No. 2 203-212
DOI: 10.1093/hmg/ddh022
A tumour-derived mutant allele of XRCC2 preferentially suppresses homologous recombination at DNA replication forks
1Institute for Cancer Studies, University of Sheffield, School of Medicine, Sheffield S10 2RX, UK, 2Department of Histopathology, Royal Hallamshire Hospital, Sheffield Teaching Hospitals NHS Trust, Sheffield S10 2UL, UK and 3Academic Unit of Pathology, University of Sheffield, School of Medicine, Sheffield S10 2RX, UK
Received October 13, 2003; Accepted November 13, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Homologous recombination repair (HRR) is required for both the repair of DNA double strand breaks (DSBs) and the maintenance of the integrity of DNA replication forks. To determine the effect of a mutant allele of the RAD51 paralog XRCC2 (342delT) found in an HRR-defective tumour cell line, 342delT was introduced into HRR proficient cells containing a recombination reporter substrate. In one set of transfectants, expression of 342delT conferred sensitivity to thymidine and mitomycin C and suppressed HRR induced at the recombination reporter by thymidine but not by DSBs. In a second set of transfectants, the expression of 342delT was accompanied by a decreased level of the full-length XRCC2. These cells were defective in the induction of HRR by either thymidine or DSBs. Thus 342delT suppresses recombination induced by thymidine in a dominant negative manner while recombination induced by DSBs appears to depend upon the level of XRCC2 as well as the expression of the mutant XRCC2 allele. These results suggest that HRR pathways responding to stalled replication forks or DSBs are genetically distinguishable. They further suggest a critical role for XRCC2 in HRR at replication forks, possibly in the loading of RAD51 onto gapped DNA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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DNA double strand beaks (DSBs) form in cells during DNA replication and following exposure to ionizing radiation or some types of DNA replication inhibitors. Cells are acutely sensitive to DSBs and as few as one or two such lesions are sufficient to trigger the activation of the ATM (ataxia telangiectasia mutated) protein kinase, one of the prime regulators of the DNA damage response (1). DSBs are repaired by two pathways. Non-homologous end joining repairs DSBs by a process that frequently results in the loss of genetic information. Homologous recombination repair (HRR) utilizes the sister chromatid to rejoin breaks in an error-free process (2). In human cells HRR is dependent upon RAD51, a functional and structural homologue of the E. coli recA recombinase, and five further proteins having sequence similarity to RAD51. Two of these RAD51 paralogues, XRCC2 and XRCC3, were isolated by their ability to correct radiosensitive hamster cell lines (3–5). The remaining three were identified from database searches on the basis of their similarity to XRCC2 and XRCC3 (6–9). Although the precise role(s) of these proteins in HRR is not clear, the functions of the five paralogues are not redundant. The loss of any one of them is sufficient to sensitize cells to agents that induce DSBs (3–5,10) and cells deficient in these paralogues show evidence of defects in homology-based recombination repair (11,12). Cells having defects in these genes have slower growth rates and high levels of spontaneous chromosome aberrations (10). Recent reports demonstrate that the human Rad51 paralogues associate into two complexes (13–18). One contains RAD51B, RAD51C, Rad51D and XRCC2 (BCDX2), while the second contains RAD51C and XRCC3. The RAD51C–XRCC3 complex was shown to bind single-stranded DNA and to promote DNA–DNA interactions resulting in DNA annealing (13,15). The BCDX2 complex binds specifically to single-stranded DNA and to single-stranded regions or nicks in duplexed DNA, consistent with the proposal that this complex may play an early role in HRR (14).
Cells deficient in HRR are particularly sensitive to agents that induce DNA crosslinks (such as mitomycin C, MMC) (3–5,10). They are also sensitive to agents inducing DSBs (such as ionizing radiation, IR, or camptothecin, CPT) and thymidine, although this agent induces few, if any, DSBs (19). We have proposed that HRR is necessary to resolve replication intermediates formed following the slowing of DNA synthesis caused by the depletion of dCTP resulting from thymidine treatment (19). This role is consistent with mounting evidence for the involvement of HRR in maintaining the integrity of collapsed or impaired DNA replication forks (20,21). We have previously presented evidence that this HRR pathway is defective in mismatch repair (MMR)-deficient tumour cell lines (22). These cells are sensitive to thymidine and are defective in the homology-directed repair of a site-specific DSB induced in a recombination reporter substrate. Thymidine sensitivity was not dependent upon the loss of MMR as cells corrected for the MMR defect remained thymidine-sensitive. Analysis of the RAD51 paralogues in the MMR-deficient tumour cell lines revealed a heterozygous mutation of XRCC2 in one (SKUT-1) derived from a uterine sarcoma. This mutant allele (342delT) contains a –1 frameshift in a run of eight thymine residues at nucleotides 342–350. Such frameshifts are characteristic of mutations occurring in MMR-deficient cells and are thought to confer a selective advantage to the tumour cells. 342 delT was predicted to encode a peptide of 132 amino acids that retains the Walker A box of the highly conserved ATPase domain but loses the equally conserved Walker B box. Preliminary work suggested that this mutant allele of XRCC2 could confer thymidine and MMC sensitivity when transfected into HRR-proficient cell lines (22). In the work presented here we analyzed the effect of this mutant allele on HRR and our data indicate that 342delT preferentially interferes with the HRR-mediated rescue of DNA replication forks stalled by thymidine.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Expression of 342delT in the MMR proficient tumour cell line SW480 containing the Scneo recombination reporter
The mutant XRCC2 allele was subcloned into the vector pIRESpuro3 to allow its introduction into SW480/SN.3 and MRC5VA/SN.13 cells containing the Scneo recombination reporter and ensure expression of the mutant allele with the puromycin resistance gene. A list of the transfectants obtained and other cell lines used in these experiments is presented in Table 1. To confirm that the transfectants expressed the mutant allele, RNA was purified from six clones obtained from the two cell lines and XRCC2 cDNA was amplified from each. The cDNAs were sequenced and were found to include both wild-type T8 and mutant T7 alleles of XRCC2 (Fig. 1A). In SW480/SN.3 transfectants both wild-type and mutant sequences were evident; however, in the MRC5VA/SN.13 transefctants, the mutant T7 allele was predominant. To ensure that these mutant sequences originated from the expression of the transgene, RNA preparations were treated with DNAse. Furthermore no product was detected in reactions using these RNA preparations when RT was omitted.
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Western blot analysis of cell free lysates prepared from the transfected cells revealed a rapidly migrating protein interacting with the XRCC2 antibody having approximately the molecular weight of the peptide predicted to be encoded by 342delT (Fig. 1B and C). This peptide was also found in SKUT-1 cells but has not been detected in any other cell line we have examined thus far. Interestingly the levels of the peptide and the full-length XRCC2 protein vary in the transfectants obtained. The SW480/SN.3 transfectants retain levels of full-length XRCC2 comparable to those found in the parental cells and show clear evidence of the presence of the mutant peptide (Fig. 1B). The MRC5VA/SN.13 transfectants show some variability in the level of the mutant peptide and, more interestingly, a reduced level of the full length XRCC2 similar to the expression pattern seen in SKUT-1 (Fig. 1C). As a result these cells have a much higher level of the mutant XRCC2 relative to the full-length XRCC2. Thus all the transfectants express the peptide encoded by 342delT; however, there is considerable variation in the level of the mutant peptide relative to the full length XRCC2 in the two cell types.
342delT confers sensitivity to thymidine and MMC
We next determined whether the strains expressing the mutant XRCC2 were sensitive to thymidine. SW480/SN.3, MRC5VA/SN.13, SKUT-1 and the transfectants expressing 342delT were plated in increasing concentrations of thymidine and allowed to form colonies (Fig. 2A and C). As reported previously, SKUT-1 cells were more sensitive to the toxic effects of thymidine than SW480/SN.3 or MRC5VA/SN.13. Consistent with our previous results (22), transfectants of SW480/SN.3 and MRC5VA/SN.13 expressing 342delT were also more sensitive to thymidine. Sensitivity to mitomycin C was determined since HRR deficient cells typically show increased sensitivity to this agent (3–5). Again SKUT-1 and the transfectants obtained from SW480/SN.3 and MRC5VA/SN.13 expressing the mutant XRCC2 allele showed increased sensitivity to this agent relative to the parental SW480/SN.3 and MRC5VA/SN.13 cells (Fig. 2B and D). MRC5VA/SN.13 cells expressing 342delT showed similar sensitivity to thymidine as SKUT-1, while the SW480/SN.3 transfectants displayed an intermediate sensitivity. This is notable as the MRC5VA/SN.13 transfectants, like SKUT-1 cells, have a depressed level of the full-length XRCC2. In contrast the SW480 transfectants maintain a level of XRCC2 similar to that found in the control cells.
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To further test the relationship between the sensitivity to thymidine and the balance of the two forms of XRCC2, an expression construct containing a wild-type XRCC2 cDNA was transfected into SKUT-1 cells to increase the level of the wild type protein. Cells containing the expression vector had a higher level of XRCC2 protein than SKUT-1 on western blots (Fig. 3A) and all of the clones expressing XRCC2 were significantly more resistant to thymidine (Fig. 3B). Taken together these data indicate that 342delT confers sensitivity to thymidine and MMC and the degree of sensitization appears to depend upon the balance between the levels of the wild type and the mutant proteins.
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342delT exerts a cell line specific effect on the repair of a site specific DSB by homology based recombination
We next determined the effect of the mutant XRCC2 on the induction of recombination. To accomplish this, we used the Scneo recombination reporter (11) contained in the parental SW480/SN.3 and MRC5VA/SN.13 strains. Homology-based recombination of the two defective G418 genes contained in this construct can be induced by the introduction of a site-specific DSB into one of the neo genes following transfection of a construct expressing the endonuclease I-SceI. Transfection of this construct into SW480/SN.3 cells led to an 850-fold increase in the frequency of neo+ colonies relative to mock transfected cultures (Fig. 4). Similarly, transfection of I-SceI into the three strains derived from SW480/SN.3 expressing 342delT resulted in a 600- to 1500-fold increase in the frequency of neo+ colonies. The induced frequencies of neo+ colonies following I-SceI transfection were not significantly different for all the cells tested.
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In contrast the frequency of neo+ colonies was not increased in MRC5VA/SN.13 strains expressing 342delT following induction of the site specific DSB (Fig. 4). Transient expression of the I-SceI endonuclease into the parental MRC5VA/SN.13 cells led to a 110-fold increase in the frequency of neo+ recombinants relative to mock transfected cultures while the strains expressing 342delT showed only a 1.7- to 2.4-fold increase in the frequency of neo+ colonies. This is similar to the suppression of recombination previously reported for SKUT-1 (22) (Fig. 4). Thus the effect of 342delT on DSB-induced recombination in the ScNeo reporter is different in the two sets of transfectants.
Cells expressing 342delT are defective in HRR induced by thymidine
Previous work has shown that cells defective in HRR become sensitive to thymidine (19). These observations indicate that thymidine treatment generates DNA lesions that must be resolved by HRR for cell survival. While the nature of these lesions is unclear, these observations suggest that the thymidine treatment should induce recombination and such effects have been reported previously (19). To examine the recombinogenic effects of thymidine in cells expressing 342delT, we treated replica cultures with increasing concentrations of thymdine and then determined the frequency of neo+ colonies. To obtain a more precise measurement of the inductive effect of thymidine, we inoculated replica cultures with 1000 cells to eliminate preexisting neo+ cells and then grew them to 
1x106 cells before treatment. Using this approach, we found that thymidine treatment increased the frequency of neo+ recombinants in parental SW480/SN.3 and MRC5VA/SN.13 cells in a dose dependent manner up to 6-fold in cultures treated with 10 mM thymidine (Fig. 5A and B).
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We next determined whether the thymidine sensitive SKUT-1 was defective in thymdine-induced recombination. Since SKUT-1 was sensitive to thymidine we were unable to treat with the highest concentrations, however following treatment with 2 mM thymidine we were unable to detect any neo+ colonies (frequency <1.8x10–7), while this concentration increases the frequency
3-fold in SW480/SN.3 (Fig. 5A). SW480SN/3 and MRC5VA/SN.13 cells expressing 342delT were then treated with thymidine to determine the effect on recombination. The SW480/SN.3 cells were treated with 2 or 10 mM thymidine since they show an intermediate sensitivity to thymidine. Following this treatment we found that the frequencies of neo+ recombinants were not increased (Fig. 5A). Similarly there was no increase in the frequency of neo+ recombinants following treatment of MRC5VA/SN.13 cells expressing 342delT with 2 or 4 mM thymidine while the parental MRC5VA/SN.13 showed a 3- to 4-fold induction at these concentrations (Fig. 5B). Thus all the cells expressing the mutant XRCC2 allele are defective in the induction of homologous recombination following treatment by thymidine.
Cells expressing 342delT are proficient for recombination induced by an agent that induces replication fork DSBs
Previous work indicated that thymidine induced few, if any, DSBs in cells, suggesting that some other DNA lesion produced by this agent is recombinogenic (19). To determine whether thymidine induced DSBs in the tumour cell lines used in these experiments, we treated cells with thymidine as well as agents known to induce DSBs. Treated cells were then harvested and analysed for DSBs by pulsed field gel electrophoresis. Consistent with our previous work, highly toxic concentrations of thymidine failed to induce detectable DSBs in parental cells or the thymidine sensitive cells expressing 342delT (Fig. 6). In contrast both IR and CPT were effective at inducing this type of damage in all the cells tested.
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Since the SW480SN3 strains expressing the mutant XRCC2 were able to produce recombinant neo genes following treatment with I-SceI it appeared that these cells were capable of repairing DSBs. Thus the deficiency of thymidine-induced recombination found in these cells could be due to a defect in the repair of lesions specifically induced by thymidine. Alternatively the altered response could be due to the level of lesions induced by these two treatments. Thymidine is potentially capable of inducing a recombination substrate at every replication fork since all are likely to be affected by this agent. In contrast a very small number of site-specific breaks are induced by I-SceI. Thus thymidine may simply saturate the HRR pathway made less efficient by the introduction of the mutant XRCC2 allele. To test this hypothesis we then determined whether camptothecin (CPT), which induces a high level of DSBs at replication forks throughout the genome (24–26) (Fig. 6) was able to induce recombination in wild type cells and cells expressing 342delT.
We first examined the sensitivity of SW480/SN.3, SKUT-1 and two transfectants expressing 342delT to CPT. SKUT-1 showed a slight, but significant increase in sensitivity to this agent while the transfectants showed intermediate sensitivity relative to the parental SW480/SN.3 (Fig. 7A). We next treated these same cells with varying concentrations of CPT to determine whether this agent induced neo+ recombinants. CPT induced neo+ recombinants in a dose dependent manner up to 10-fold in SW480/SN.3 (Fig. 7B). The two transfectants expressing 342delT, showed similar levels of neo+ recombinants at all concentrations of CPT. Thus SW480/SN.3 cells expressing 342delT are able to induce recombination following CPT treatment but not thymidine.
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Detection of the 342delT mutation in uterine leiomyosarcomas
Given that the tumour cell line in which we detected 342delT was derived from a uterine sarcoma, we determined whether the 342delT mutation is common in such tumours. DNA was extracted from archival tissue blocks of eight uterine leiomyosarcomas and the XRCC2 exon containing nt 342 was amplified from this DNA and sequenced. The 342delT frameshift was found in one of these tumours (a high-grade uterine leiomyosarcoma) so the mutation can be detected in primary tumour tissue. Adjacent normal myometrium in this case showed no mutation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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342 delT interferes with thymidine-induced HRR in a dominant negative manner
The original objective of this work was to determine whether the mutant allele of XRCC2 found in the uterine sarcoma cell line SKUT-1 was responsible for the deficiency in homology-directed DSB repair we found in that cell line. The results reported here support our hypothesis although the phenotypic effects in the transfectants appear to depend upon the levels of the mutant and wild type proteins. Our data suggest that 342delT acts as a dominant negative to suppress HRR induced by thymidine. This suppression of thymidine-induced recombination is accompanied by increased sensitivity to thymidine and MMC. In contrast the effect of 342delT on DSB-induced recombination following I-SceI or camptothecin treatment is more complex. In SW480/SN.3 transfectants levels of the full-length XRCC2 are similar to those found in parental cells and the mutant allele is clearly expressed. Thus suppression of thymidine-induced recombination in the SW480/SN.3 transfectants is not the result of XRCC2 haploinsufficiency. Instead the phenotype displayed by the SW480/SN.3 transfectants appears to depend upon the expression of the mutant XRCC2 gene. The more extreme phenotype exhibited by the MRC5VA/SN.13 transfectants expressing 342delT and SKUT-1 appears to be the result of both the expression of the mutant allele and the depression of the level of the wild-type XRCC2. Consistent with this suggestion is our observation that SKUT-1 cells, in which the level of the wild-type XRCC2 was increased by the transfection of a construct expressing the wild-type cDNA, became more resistant to thymidine.
The mechanism responsible for the depression of the level of the full-length XRCC2 protein in MRC5VA/SN.13 cells and SKUT-1 is not clear. MRC5VA/SN.13 transfectants appear to produce an excess of the mutant transcript as judged by the level of the two types of cDNAs amplified from these cells; however, it is unlikely that enhanced expression of the mutant allele would suppress expression of the wild-type. Nonsense-mediated decay, which alters the stability of messenger RNAs containing nonsense mutations, should specifically affect the mutant gene in these cells, not the wild type (for review see 27). One possible explanation is that the decreased level of XRCC2 is the result of increased turnover of the protein. It is known that XRCC2 forms a complex with other RAD51 paralogs (13,17). The mutant peptide may associate more effectively with the complex than the full-length protein and the uncomplexed protein may be degraded. This would be similar to the situation with mismatch repair proteins hMLH1 and hPMS2, which are normally found in cells in the form of heterodimers (28). Nonsense mutations that eliminate the full-length wild-type hMLH1 also result in a decline in the level of the uncomplexed partner protein hPMS2 (29).
XRCC2 342delT mutant protein preferentially impairs repair at stalled replication forks
Here we show that thymidine does not induce HRR in SW480/SN.3 cells expressing 342delT (Fig. 5A). However, these cells maintain the ability to utilize homologous recombination to repair DSBs induced by either I-SceI or camptothecin. We propose that this altered HRR capacity is likely to reflect the different lesion formed following thymidine treatment, which may not include a DSB (Fig. 7) (19). Long tracts of gapped DNA form at stalled forks in wild-type yeast (30). In E. coli, the RecFOR complex loads the RecA protein onto gapped DNA sequences, which suggests that such regions may initiate HRR (31). The purified human RAD51 paralogue complex, which includes XRCC2, colocalizes with RAD51 on gapped DNA substrates, suggesting a role for this complex in the stabilization or recruitment of RAD51 nucleoprotein filaments (14). For invasion to occur RAD51 must replace the RPA protein on the ssDNA gap, which might not be easy, given that RPA has a greater affinity for ssDNA than RAD51. Replacement of RPA with RAD51 may be catalysed by the RAD51 paralogues (32). This is a very interesting observation suggesting that strand invasion can be initiated without a free DNA end. Taken together these reports suggest that gapped DNA may initiate HRR and that this process requires a functional XRCC2 protein.
The presence of gapped DNA at stalled replication forks in yeast raises the possibility that replication forks stalled by thymidine in human cells include gapped DNA regions. We suggest that the mutant XRCC2 allele may preferentially interfere with thymidine-induced recombination in SW480/SN.3 cells by impairing the loading of RAD51 onto gapped DNA sequences. However, this mutant allele may not interfere with the repair of DSBs as strongly and the relatively high level of wild-type XRCC2 protein in these cells may be sufficient to load RAD51 onto DNA ends at a DSB. SKUT-1 cells and the MRC5VA/SN.13 transfectants are deficient in HRR induced by thymidine or DSBs. We believe this observation is explained by the low level of wild-type XRCC2 protein in these cells, which is known to impair the HRR of DSBs (11). Thus, our data suggest that the presence of 342delT is sufficient to cause a deficiency in HRR of thymidine-induced damage. A general deficiency in HRR is only obtained in those cells that also show decreased levels of the wild type XRCC2 protein. Thus our results suggest that it is possible to genetically distinguish different pathways for HRR in mammalian cells.
Defects in HRR gynaecological tumours
While 342delT appears to account for the HRR defect reported in SKUT-1, such mutations do not appear to be common in MSI+ or microsatellite stable tumours. This mutant gene was not detected in other MMR deficient, MSI+ tumour cell lines or a collection of 16 MSI+ and 92 microsatellite stable colon cancers (J. Scorah and M. Meuth, manuscript in preparation). SKUT-1 was derived from a uterine sarcoma (23) and examination of a collection of eight of these rare tumours revealed one that carried the same XRCC2 mutation. While this particular mutation was found in only one tumour, a translocation breakpoint affecting the last exon of a uterine specific isoform of RAD51B has been found in a subset of the more common uterine leiomyomas and results in the production of a truncated form of this RAD51 paralogue (33). Thus disruptions of this HRR pathway may be more common in some types of gynaecological mesenchymal neoplasms. The intriguing question is what advantage the loss of this repair pathway confers to developing tumours. We suggest this is likely to relate to the recently reported role of XRCC3 and HRR in the delay of DNA replication fork progression following DNA damage (34). Cells losing HRR function through mutations of XRCC2 may suffer deregulation of DNA synthesis on damaged templates, leading to higher levels of genetic instability and indeed such effects in XRCC2 deficient cells have been demonstrated (35). Studies from our group have shown that this HRR pathway is defective in MSI+ colon cancer cell lines, although different alterations appear to account for this effect. Further work is underway to determine how commonly this repair pathway is altered in tumours and the genetic alterations underlying this disruption.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell lines and culture
The MMR-proficient colorectal carcinoma cell line SW480 and MMR-deficient uterine sarcoma cell line Skut-1 were obtained from American Type Culture Collection (Manassas, VA, USA). The immortalized human diploid fibroblast cell line, MRC5VA, was obtained from Coriell. All cell lines were cultured as monolayers in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum in a humidified 5% carbon-dioxide incubator, at 37°C. Cells stably transfected with the ScNeo construct were maintained in medium supplemented with Hygromycin (final concentration, 0.525 M).
Cytotoxicity assays
The cytotoxic response to MMC and CPT were measured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum. The cytotoxic response to thymidine was measured in medium supplemented with 10% dialysed serum (to remove exogenous sources of deoxynucleosides). Aliquots of 500–1000 cells were plated into 100 mm culture dishes and treated with varying concentrations of thymidine, camptothecin and mitomycin C. Cells were allowed to grow for 10–14 days in a humidified 5% carbon-dioxide incubator, at 37°C, before staining with 0.4% methylene blue/50% methanol (Fisher Scientific). Colonies of >50 cells were scored. The surviving fraction was determined by dividing the average number of colonies for each treatment by the average number of colonies in the control wells. All experiments were plated in triplicate and were repeated independently two to five times.
Plasmid construction and transfection
The mutant XRCC2 allele (342delT) was subcloned into the pIRESpuro3 vector (Clontech) by subcloning it from the previously reported pcDNAXRCC2m vector (22). The inserted XRCC2 cDNA was sequenced, as previously described, to confirm the presence of 342delT. The pIRES-XRCC2m vector was transfected into the SW480/SN.3 and MRC5VA/SN13 MMR-proficient cell lines using Lipofectamine (Invitrogen) according to the manufacturer's protocol. Six colonies were isolated and expanded in puromycin-selective medium (0.75 µg/ml, final concentration). To verify expression of the mutated allele, mRNA was extracted from each of the six clones using the RNeasy Protect Mini Kit (Qiagen), and the XRCC2 cDNA was sequenced.
Western blot analysis
Growing cultures were used to prepare cell extracts using RIPA buffer in the presence of the proteinase inhibitor PMSF (100 µg/ml). A 50 µg aliquot of each protein sample was size fractionated on 12% sodium dodecyl sulfate polyacrylamide gels and transferred to nitrocellulose membrane (Immun-blot PVDF, Bio-Rad) using a Trans-Blot® Semi-dry Transfer cell (Bio-Rad). Membranes were blocked with 5% milk for 1 h, and the XRCC2 protein was detected using a goat polyclonal antibody (Santa Cruz Biotechnologies) at a 1 : 200 dilution in 3% milk overnight. Anti-goat peroxidase conjugates (Santa Cruz Biotechnologies) were used as a secondary antibody at a dilution of 1 : 10 000. Immunoreactive protein was visualised using the Amersham Enhanced Chemiluminescence (ECL) system according to manufacturer instructions.
Recombination assays
The ScNeo recombination reporter and the pCMV3nls-I-SceI expression vector were kind gifts from Dr Maria Jasin, Memorial Sloan Kettering Cancer Center. Strains of MMR-proficient (SW480/SN.3 and MRC5VA/SN.13) and -deficient (Skut-1/SN.3) tumour cell lines carrying single copies of the ScNeo recombination reporter were obtained as described previously (22). To measure recombination events induced by a double-strand break, cells were transfected with 10 ng of the pCMV3nls–I-SceI expression vector using Lipofectamine (5 h treatment, with a final concentration 10 µg/ml; Invitrogen). To measure recombination events induced by thymidine or CPT, replica cultures were inoculated with 1000 cells to reduce the level of spontaneous recombinants. Replica cultures were then grown to 106 cells before treatment with varying doses of thymidine or CPT. For each experiment six replica cultures were treated with thymidine or CPT and an additional six replica cultures were mock treated as controls. Following treatment the replica cultures were allowed to recover in complete medium for 48 h. Treated and control cultures were then plated in selection medium containing 1 mg/ml G418 at a cell density of 13–25 cells/mm2. In addition, two dishes were plated with 500 cells each to measure cloning efficiency. Cells were allowed to grow for 12–14 days before staining with 0.4% methylene blue/50% methanol (Sigma). Only colonies of 
50 cells were subsequently scored. The frequencies of neo+ recombinants presented are the number of neo+ colonies formed/(cells platedxthe cloning efficiency of the plated cultures). All experiments were repeated independently two to four times.
Pulse-field gel electrophoresis
Plates were inoculated with 5x106 cells 24 h prior to treating cells for 24 h with thymidine (10 mM) or CPT (100 nM), the cells were then trypsinised and 1x106 cells were melted into agarose inserts. For ionising radiation treatment 1x106 cells were melted into an agarose insert and then treated with 10 Gy of IR. The agarose inserts were then incubated in 0.5 M EDTA, 1% N-laurylsarcosyl and proteinase K (1 mg/ml) for 48 h then washed four times in TE buffer, prior to loading onto a 1% agarose gel. Pulse-field gel electrophoresis (Biorad: 120° angle, 60–240 s switch time, 4 V/cm) was then carried out. The gel was subsequently stained with ethidium bromide and analysed using Image Gauge software.
Analysis of gynaecological tumours
Appropriate formalin fixed, paraffin embedded tissue blocks were selected from eight cases of uterine leiomyosarcoma. Normal myometrium and tumour tissue were separately marked by the histopathologist (J.S.) after which 10 µm sections were microdissected to give enriched (>80%) normal and tumour tissue. DNA was extracted, following digestion with Proteinase K, using the QIAamp® (Qiagen, UK) kit, following the manufacturer's protocol. A fragment of Exon 3 (including the T8 run) of XRCC2 was PCR amplified using 0.4 µM of the primer pair XRCC2F (5'-TGATATGCTCCGGCTAGTT-3') and XRCC2R (5'-AGTTCACACTTTCTCCTCCA-3'). The PCR cycle was 94°C, 3 min, (94°C, 30 s; 52°C, 45 s; 72°C, 1 min)40, 72°C, 5 min.
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ACKNOWLEDGEMENTS |
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This work was supported by a program grant from Yorkshire Cancer Research to M.M. and a PhD studentship to A.M. from the P.A. Banner Trust. We are grateful to Jarek Dziegielewski for his helpful suggestions concerning these experiments.
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FOOTNOTES |
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* To whom correspondence should be addressed at: Institute for Cancer Studies, The University of Sheffield School of Medicine, Beech Hill Road, Sheffield S10 2RX, UK. Tel: +44 1142713288; Fax: +44 1142713515; Email: m.meuth{at}sheffield.ac.uk
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