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[Cancer Research 63, 8181-8187, December 1, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Homologous Recombination Deficiency Leads to Profound Genetic Instability in Cells Derived from Xrcc2-Knockout Mice

Bryan Deans1, Carol S. Griffin1, Paul O’Regan1, Maria Jasin2 and John Thacker1

1 Medical Research Council, Radiation and Genome Stability Unit, Harwell, Oxfordshire, United Kingdom, and
2 Sloan-Kettering Cancer Center, New York, New York


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA damage such as double-strand breaks presents severe difficulties for the cell to repair, especially if genetic stability is to be preserved. Recombination of the damaged DNA molecule with an undamaged homologous sequence provides a potential mechanism for the high-fidelity repair of such damage, and genes encoding homologous recombination (HR) proteins have been identified in mammalian cells. Xrcc2 is a protein with homology to Rad51, the core component of HR, but with a nonredundant role in damage repair. Here, we make the first study of the consequences of knocking out one or both copies of the Xrcc2 gene in mouse cells. In addition to growth arrest and sensitivity to agents causing severe DNA damage, we show that order-of-magnitude higher levels of chromosomal alterations are sustained in primary or immortal Xrcc2-/- embryonic fibroblasts. Using spectral karyotyping, we find that aneuploidy and complex chromosome exchanges, including an unexpectedly high frequency of homologue exchanges, are hallmarks of Xrcc2 deficiency. In addition, we find evidence for mild haploinsufficiency of Xrcc2. These responses are linked to several indicators of reduced HR in Xrcc2-/- cells, including a 30-fold reduction in gene conversion and reduced levels of Rad51-focus formation and of sister-chromatid exchange. Our data have similarities to recent studies of the disruption of breast cancer-predisposing (Brca) genes in mouse cells and are contrasted to analyses of cells carrying disruptions of genes in the other main pathway for double-strand break repair, nonhomologous end joining.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The faithful repair of DNA damage is important for the survival of cells and to maintain genetic stability. To repair severe forms of DNA damage such as DSBs,3 two major pathways have evolved. HR uses an undamaged homologous sequence as a template for repair, whereas NHEJ simply ligates broken DNA ends, with potential loss of sequence integrity (1) .

Mammalian cells show structural and functional conservation of key HR proteins such as Rad51 (2) . In addition, several Rad51-like proteins have been identified in mammalian cells, although these are less well conserved than Rad51 itself (3) . The Rad51-like proteins have been shown to form specific complexes that appear to assist Rad51 in the strand exchange mechanism that is central to recombination (4 , 5) . Despite the presence of five Rad51-like proteins in mammals, namely Xrcc2, Xrcc3, Rad51L1, Rad51L2 (Rad51C), and Rad51L3, these do not show functional redundancy. For example, targeted deletion of the Xrcc2 gene alone in mice gives embryonic lethality and severe developmental defects (6) . However, the cellular roles of the mammalian Rad51-like proteins are still relatively unknown, especially in safeguarding genetic stability. Indeed, relatively little detailed information has been published on the nature of genetic changes occurring in mammalian HR-deficient cells (7 , 8) in contrast to a number of recent studies of NHEJ-deficient mouse knockout cells (9, 10, 11, 12, 13) . Importantly, mutations in the Brca genes, which predispose to breast, ovarian, and other cancer types, lead to reduced HR (14, 15, 16, 17) through disruption of interaction of Brca2 with Rad51 (18 , 19) and to a variety of types of chromosomal changes, including aneuploidy, translocations, and deletions (20 , 21) . It can be inferred therefore that loss of Rad51 activity leads to serious genetic instability, and indeed, switching off Rad51 expression in chick cells results in their accumulation in G2-M phase with large amounts of chromosomal breakage and cell death (22) .

We now report a detailed comparison of the responses of isogenic mouse embryonic cells carrying a targeted deletion of one or both copies of the Xrcc2 HR gene with their wild-type counterparts. Using these Xrcc2-deficient cells, we have been able to assess for the first time the possibility of haploinsufficiency for a number of responses, as well as the first detailed analysis of chromosomal instability in Xrcc2-deficient cells using fluorescence-based techniques, including SKY. We show that loss of Xrcc2 results in growth arrest, sensitivity to DNA-damaging agents, recombination defects, and an extraordinary level of chromosomal instability.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Culture of MEFs and ES Cells.
Fibroblasts were isolated from 13.5 d embryos from Xrcc2+/- mouse crosses (6) . Embryo tissue was disrupted and MEFs cultured in DMEM with 10% fetal bovine serum, 2-mercaptoethanol (100 µM), and antibiotics. Genotyping was by PCR (6) . Cellular proliferation was assayed by plating MEFs at 8 x 104 cells/3.5-cm plate over a period of 10 days. Flow cytometric analysis was carried out on exponentially growing passage 1 cultures using a FACsort (Becton Dickinson). Xrcc2+/+ and Xrcc2+/- embryonic fibroblast cell lines were established by 3T3-equivalent culture as described previously (23) . Xrcc2-/- cultures were grown to passage 3 and then maintained at high density with regular feeding. Passaging was recommenced when mitotic cell foci were seen at ~4–6 weeks. Clones that had overcome crisis were used at passages 14–20. ES cells were isolated from 3.5-day blastocysts derived from crosses of Xrcc2+/- mice as previously described (24) and were maintained in the presence of mitotically inactivated (30 Gy X-irradiated) primary MEFs using growth media as above with added ESGRO (Chemicon). Xrcc2-/- ES cell cultures proliferated initially less well than wild-type cultures but improved with passage. Dominant negative p53 constructs (25) were kindly supplied by Ashok Venkitaraman, Hutchison/MRC Research Centre (Cambridge, United Kingdom; Ref. 26 ).

Mouse Xrcc2 cDNA Cloning and Complementation of Xrcc2-/- Cells.
MmXrcc2 cDNA was isolated from mouse embryonic tissue RNA and cloned into the bicistronic expression vector pIREShyg (Clontech). MEFs were transfected using Fugene (Roche Diagnostics) as recommended and were selected in 100–250 µg/ml hygromycin (Calbiochem). All hygromycin-resistant clones picked showed good functional complementation, as seen by MMC resistance (10 of 10 for MEFs, 12 of 12 for ES cells), and pools of these clones were used in additional experiments.

MMC and {gamma}-Irradiation Survival Assays.
Exponentially growing immortal cells were treated with MMC (0–5 µM) for 2 h in DMEM without FBS at 37°C. Cells were then washed twice with PBS, trypsinized, and counted before dilution and plating. For {gamma}-irradiation survival assays, exponentially growing cells were trypsinized and irradiated with doses of up to 10 Gy (60Co, 1.1 Gy/min). Treated cells were respread with feeder cells (8 x 104 cells for MEFs, 5 x 105 for ES cells in 6-cm dishes). Colonies were counted after 9–14 days incubation and survival expressed relative to untreated control populations.

Recombination Assay and Targeted Integration.
ES cells were targeted with p59xDR-GFP recombination substrate at the Pim-1 locus and assayed for HR repair proficiency (15) . Targeted ES cells were identified by Southern analysis (15) or by PCR with the following primers: sense primer to Pim-1 gene 5' to region of targeting, 5'-ATGCTCCTGTCCAAGATCAAC-3'; antisense primer to the adjacent hygromycin gene, 5'-CGGTGAGTTCAGGCTTTTGAG-3' to give a 600-bp product. Exponentially growing targeted cells were electroporated (250 V, 960 uF) with I-SceI expression vector (pCBASceI, 50 µg), and the frequency of gene-converted GFP-positive cells was determined by flow cytometry after 48 h of culture and compared with vector-only controls. Frequencies of recombination were corrected for electroporation efficiency as determined by electroporation of control populations with GFP expression vector (pNZE-CAG).

Rad51 Immunofluorescence.
Immortal MEFs were analyzed 5 h after 10 Gy X-rays as previously described (27) , using anti-hRAD51 antibody (Ab-1; Oncogene Research at 1:100 dilution). Confocal microscopy images (50 cells/data point) were scored blind to record the number of discrete strongly fluorescing nuclear foci present in each cell.

Cytogenetics.
Primary MEFs from 14-day embryos were incubated overnight and then exposed to Colcemid for 2 h before harvesting for metaphase preparation. Immortal MEFs were grown to confluence to give a majority of the cells in G1, and metaphases were collected after 4 h in Colcemid. A significant proportion of the immortal MEFs showed hyperploidy, and aberrations were scored in cells with <65 chromosomes. To make accurate comparisons, aberration frequencies in immortal cells are given per chromosome. FISH was carried out using FITC and/or biotin-labeled chromosome-specific probes for chromosomes 2, 4, and 11 (Cambio) together with a cy3-labeled pancentromeric probe. Biotin was detected using Texas red- conjugated streptavidin. FISH was carried out as previously described (28) , and patterns were classified according to Savage and Simpson (29) . SKY procedures were as given in the SkyPaint hybridization and detection protocol (Applied Spectral Imaging). Pretreatment consisted of RNase A (100 µg/ml) for 1 h at 37°C and pepsin for 2 min at 20°C (50 µg/ml in 10 mM HCl; Sigma), with counterstaining by DAPI. To measure SCEs, BrdUrd (5 µg/ml) was added to the cells for 24 h (approximately two cell cycles) before harvesting to differentially label the chromatids, followed by Hoechst 33258/UV treatment and Giemsa staining to obtain dark (TdR/BrdUrd) and pale (BrdUrd/BrdUrd) chromatids. Approximately 100 cells were scored per genotype. Centrosomes and microtubules were detected in passage 1 MEFs grown on glass slides, as previously described (30) using anti-{gamma}-tubulin (Sigma) and anti-ß-tubulin (Sigma), respectively. Approximately 150 mitotic cells were scored per genotype.

Calculations and Statistics.
To calculate the expected frequency of rearrangements between homologous chromosomes, each mouse chromosome was assumed to break and rejoin at random, with allowance for the relative length of each chromosome. Calculating the sum of squares of the relative chromosome lengths, the proportion of exchanges that are between homologues was found to be 2.83% (D. Papworth, personal communication). Significance of the experimental data was calculated using ANOVA tests, using the Mann-Whitney U test for comparisons of significance.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Primary Embryonic Fibroblasts (MEFs) from Xrcc2-/- Mice Show Rapid Growth Arrest.
We have previously reported the disruption of Xrcc2 in mice in which deletion of exon III (encoding 86% of the protein) was produced by gene targeting (6) . In contrast to Xrcc2+/+ (n = 10 embryos) and Xrcc2+/- (n = 32) MEFs, Xrcc2-/- (n = 9) MEFs underwent rapid proliferative arrest (Fig. 1A)Citation with accumulation in G2 (wild-type 14%, Xrcc2-/- 38%) and a marked increase in hyperploidy (Fig. 1B)Citation . Proliferation had largely ceased by passage 3 in Xrcc2-/- cultures, whereas Xrcc2+/+ and Xrcc2+/- cells remained highly replicative up to passages 7–8, as described for wild-type mouse cell cultures (23) .



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  		Fig. 1. Early passage Xrcc2-/- MEFs fail to proliferate. A, growth curves at passages 2 and 3; B, FACs profiles at passage 1 (representative data).

 
Replicative arrest of Xrcc2-/- cells was accompanied by morphological characteristics that reflected cellular senescence, which may reflect the triggering of damage checkpoints by unrepaired or misrepaired DNA damage. In support of this, arrest was overcome in the presence of dominant negative forms of p53 (G154V or R273L; data not shown). Furthermore, we have been able to isolate immortal Xrcc2-/- cells that spontaneously overcame senescence. Unlike Xrcc2+/+ and Xrcc2+/- cells, immortalization did not occur for Xrcc2-/- cells during 3T3-equivalent culture because their numbers dwindled under this regimen. However, in cultures that were expanded until passage 3 and then maintained at high density, foci of proliferating Xrcc2-/- cells were evident after a period of 4–6 weeks (at a frequency of ~1 focus/106 cells). These immortal cells have been used to validate primary cell studies and were also used where premature senescence limited or negated the use of Xrcc2-/- primary cultures.

Xrcc2 Affects {gamma}-Ray Resistance Differentially in MEFs and ES Cells.
Using immortal MEFs, it was found that Xrcc2-/- cells showed a reproducible small enhancement in sensitivity to {gamma}-rays (1.3-fold), whereas Xrcc2+/- cells were indistinguishable from wild-type cells (Fig. 2A)Citation . Xrcc2-/- MEFs were considerably more sensitive to the DNA cross-linking agent MMC than wild-type cells (4.5-fold), but again Xrcc2+/- MEFs were as resistant as wild-type MEFs (Fig. 2B)Citation . ES cells derived from the different Xrcc2 genotypes (see "Materials and Methods") were similarly tested (Fig. 2, C and D)Citation . Xrcc2-/- ES cells were noticeably more sensitive to {gamma}-rays than MEFs (2-fold), but sensitivity to MMC was similar to that for MEFs (5-fold). Xrcc2+/- ES cells were again indistinguishable from wild-type cells. To verify that sensitivity differences related specifically to Xrcc2, we also measured survival in Xrcc2-/- clones transfected with mouse Xrcc2 cDNA. The cDNA transfectants had wild-type resistance to MMC for both MEFs and ES cells and for ES cells treated with {gamma}-rays. However, for MEFs, the Xrcc2-/- cDNA gave greater-than-wild type resistance to {gamma}-rays, perhaps reflecting their relatively small radiation sensitivity (Fig. 2)Citation .



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  		Fig. 2. Xrcc2-/- embryonic cells are hypersensitive to {gamma}-rays and MMC, and this defect can be corrected by mouse Xrcc2 cDNA. Points on curves are means ± SE of data from two to three experiments/cell culture with cells derived from two embryos (immortal MEFs) or one embryo (ES cells). A, {gamma}-rays, immortal MEFs; B, MMC, immortal MEFs; C, {gamma}-rays, ES cells; and D, MMC, ES cells.

 
Reduction in Gene Targeting and Intergenic Recombination at a DSB in Xrcc2-/- ES Cells.
To assess the molecular basis for DNA damage sensitivity, ES cells representing the different Xrcc2 genotypes were transfected with a recombination reporter construct (p59xDR-GFP6) designed to target a specific site in the genome (the Pim1 gene on chromosome 17; Ref. 15 ). Gene targeting was highly efficient in Xrcc2+/+ and Xrcc2+/- ES cells with 100% (five of five) Xrcc2+/+ clones and 86% (six of seven) Xrcc2+/- clones that survived hygromycin selection showing correct recombination at the targeted locus. The efficient targeting obtained with this vector is consistent with previous results in which 97% of selected clones were gene targeted (15 , 31) . However, in parallel experiments, targeting of Xrcc2-/- ES cells was highly inefficient with only 1 in 196 (~0.5%) of correctly targeted clones. The reporter construct carries two mutant GFP genes; one of the GFP genes is modified to incorporate an I-SceI endonuclease site, allowing a DSB to be generated within the gene, whereas the other is truncated at both ends. After induction of a DSB at the I-SceI site, recombination (gene conversion to give an active GFP gene) can be measured by fluorescence-activated cell sorting. The recombination frequency in the Xrcc2+/+ cells was ~4%, similar to previous data for wild-type mammalian cells (15 , 32) . However, there was a dramatic reduction in the recombination frequency for the Xrcc2-/- ES cells (~30-fold; P = 10-5 relative to wild type). This defect was largely restored by introduction of the Xrcc2 gene (Fig. 3A)Citation , showing again that the defect was gene specific (not significantly different from wild type; P = 0.20). It was of interest that the heterozygous Xrcc2+/- cells also showed a modest reduction in recombination frequency (1.5-fold; not significantly different from wild type; P = 0.11).



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  		Fig. 3. Loss of Xrcc2 compromises HR. A, recombination in Xrcc2-deficient ES cells, measured by homology-directed repair (gene conversion) of a DSB gene in an inactive GFP gene (means ± SE from three independently targeted clones for each of Xrcc2+/+, Xrcc2+/-, and complemented Xrcc2-/- genotypes and from one clone for Xrcc2-/-). B, loss of Rad51 focus formation in immortal Xrcc2-/- MEFs. SEs are shown from means of two to three experiments/cell line.

 
Xrcc2 Is Required for SCE and Rad51 Focus Formation.
It is anticipated on the basis of current data (33 , 34) that recombination-deficient cell lines will show a reduction in the frequency of SCEs. However, the Xrcc2-deficient hamster cell line, irs1, shows a higher frequency of SCEs than wild-type hamster cells, even after treatment with DNA damaging agents (Ref. 35 and unpublished data). To address this anomaly, we measured SCE frequency in both primary and immortal Xrcc2-/- MEFs, relative to the isogenic wild-type Xrcc2+/+ cells. We found in both cell types that the SCE frequency was reduced by one-third in Xrcc2-/- cells (primary cells, 0.16 versus 0.24 (P = 0.002); immortal cells, 0.24 versus 0.35 (P = 0.0004) SCE/chromosome for Xrcc2-/- versus Xrcc2+/+ cells). To further implicate mouse Xrcc2 in Rad51-dependent recombination processes, we measured the formation of Rad51 foci in immortal Xrcc2-/- MEFs after 10 Gy X-irradiation (Fig. 3B)Citation . In wild-type MEFs, irradiation gave a large increase in Rad51 foci (P = 10-6 compared with unirradiated cells), whereas Xrcc2-/- MEFs showed no significant increase (P = 0.09). Xrcc2-/- MEFs transfected with the mouse Xrcc2 cDNA gave a level of radiation-induced foci indistinguishable from wild-type cells (P = 0.24). Even in unirradiated cells, there was a slight but significant reduction in focus formation for the Xrcc2-/- MEFs compared with wild type (P = 0.01), but this was also true for the cDNA-complemented cells (P = 0.021).

Xrcc2-Deficient MEFs Are Genetically Unstable.
We wished to see whether the recombination deficiency in the Xrcc2-knockout mouse cells would lead to genetic instability. Despite growth difficulties it was possible to score chromosomal damage in very early passage MEFs. Using three-color FISH, first passage Xrcc2-/- MEFs showed a dramatic increase in the total aberration frequency, but importantly, there was also a small increase in aberration frequency for Xrcc2+/- heterozygotes compared with wild-type MEFs (Fig. 4A)Citation . In both Xrcc2-/- and Xrcc2+/- cells, this increase consisted particularly in interchromosomal rearrangements (>4:1 rearrangements:fragments), and 16% of these were complex in Xrcc2-/- cells (Table 1)Citation . Detached centromeres were also a significant aberration; in early passage cultures, these were only seen in the in the Xrcc2-/- MEFs. In immortal MEFs, the differences between genotypes were substantiated, and a massive 100-fold increase in total aberration frequency was found for the Xrcc2-/- MEFs (Fig. 4A)Citation . Again, in the immortal MEFs, these differences mainly reflected an increase in chromosomal rearrangements rather than fragments. Also detached centromeres were ~10 times more frequent in the null cells and about twice as frequent in the heterozygotes, compared with wild-type cells (Table 1)Citation . In separate experiments, we also looked for interactions that may promote specific forms of exchange such as telomere-telomere fusions or the intrachromosomal fusion of sister chromatids but did not find an excess of these in any of the genotypes (data not shown). Chromosome aberration induction is a classic response to ionizing radiations, and it was found that frequencies of rearrangements were substantially increased in wild-type and heterozygous cells after 2 Gy X-rays (Table 1)Citation . Interestingly, however, there was little or no such increase for Xrcc2-/- cells, reflecting the very high levels of spontaneous rearrangements.



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  		Fig. 4. Genetic instability in Xrcc2-/- MEFs. A, chromosome aberration frequencies revealed by three-color FISH of primary, immortal, and X-irradiated immortal cells; B, examples of homologue-homologue rearrangements in primary Xrcc2-/- MEFs revealed by SKY (top: 5/5 dicentric; bottom, 1/1 non-sister exchange, with each image shown as: true color/DAPI/pseudocolour); C, SKY karyotype of primary Xrcc2-/- MEF showing loss and gain of chromosomes (1 x 10, 3 x 18, and 0 x 17) and a complex aberration (bottom), with each chromosome shown as DAPI/pseudocolor; D, involvement of different chromosomes in loss and gain in primary Xrcc2-/- MEFs (SKY data). Each chromosome is indicated by a number, and its loss (below) or gain (above) shown as a vertical bar.

 

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  		Table 1 Frequencies of chromosome aberrations in Xrcc2-/- MEFs (three-color FISH)

 
Loss of Xrcc2 Promotes Rearrangements between Homologous Chromosomes.
To follow up our observations of a large excess of rearrangements in the Xrcc2-/- cell cultures, we used SKY. SKY allows each chromosome to be differentially labeled and examined for specific types of rearrangement. In very early passage primary MEFs, SKY analysis of the Xrcc2+/+, Xrcc2+/-, and Xrcc2-/- cultures showed a similar overall frequency of chromosome rearrangements, including the ratio of simple:complex exchanges, as found with three-color FISH (Table 2)Citation . Surprisingly, however, an elevated frequency of rearrangements between homologous chromosomes was seen in the Xrcc2-/- MEFs (examples in Fig. 4BCitation ). It can be calculated ("Materials and Methods") that ~3% of exchanges between randomly distributed chromosomes will involve homologues, whereas our data showed 21% (8 of 38) of rearrangements involved homologues in Xrcc2-/- MEFs. Rearrangements between heterologous chromosomes were mainly complex and nonreciprocal, as seen in Fig. 4CCitation . Again, SKY analysis reveals detached centromeres only in the Xrcc2-/- primary MEFs, not in the other genotypes, supporting the overall data shown in Table 1Citation .


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  		Table 2 SKY of primary MEFsa

 
Aneuploidy and Centrosome Defects in Xrcc2-Deficient Cells.
Extensive chromosome number changes were seen in the primary Xrcc2-/- MEFs using SKY (Table 2Citation ; Fig. 4CCitation ). More than 90% of Xrcc2-/- MEFs showed chromosome number changes, compared with only 5% in the wild-type MEFs. As shown in Fig. 4DCitation , most chromosomes were involved in both loss and gain; the largest chromosomes showed few losses, possibly consistent with their large gene content. We have previously shown that Xrcc2-deficient hamster cells have an increase in the frequency of centrosome fragmentation at mitosis and suggested that this may be linked to nondisjunction and aneuploidy (30) . To check this in our isogenic series of primary MEFs, we probed diploid mitotic cells with antibodies to {gamma}-tubulin, which forms the core of centrosomes. In Fig. 5Citation , it is seen that centrosome fragmentation was increased in Xrcc2-/- MEFs by ~5-fold (P = 0.005) but was also significantly increased in Xrcc2+/-cells compared with wild type (P = 0.04).



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  		Fig. 5. Centrosome fragmentation in Xrcc2-deficient cells. Mean counts from passage 1 primary MEFs derived from three to four embryos/genotype, with SEs.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Loss of Xrcc2 results in a very large reduction in HR capacity, as measured by the frequency of gene targeting and by homology-directed DSB repair. Furthermore, we show that the Xrcc2-deficient MEFs lose ability to form damage-dependent Rad51 nuclear foci and have a reduction in the frequency of SCE. These data support and extend earlier work with the Xrcc2-deficient hamster line irs1 (27 , 36) and with DT40 chick xrcc2-deficient cells (37) , showing that Xrcc2 is important for Rad51-dependent HR processing. However, the use of the mouse knockout cells has led to several novel observations on the role of Xrcc2 in cellular responses.

DNA Damage Sensitivity in Xrcc2-Knockout Cells.
We have shown previously that Xrcc2 deficiency in mice leads to embryonic lethality, although some embryos can survive almost to term with evidence of growth retardation and abnormality (6) . We now show that primary fibroblasts derived from these embryos have arrested growth. The arrest is likely to arise from a build-up of DNA damage in the absence of Xrcc2, triggering cell cycle checkpoints. In support of this interpretation, we show that even at early passage the Xrcc2-/- cells accumulate in G2 phase and that dominant negative expression of the cell cycle control/tumor suppressor protein p53 abrogates the growth arrest.

Loss of Xrcc2 also gives hypersensitivity to DNA damaging agents such as {gamma}-rays and MMC. Interestingly, the response to {gamma}-rays varies between Xrcc2-/- MEFs and ES cells, with ES cells being the most sensitive, whereas the response to MMC varies little with cell type. Severe forms of radiation damage such as DSB can be repaired by either NHEJ or HR, whereas MMC damage is repaired primarily by HR (38) . Therefore, the differences in sensitivity to these agents can be explained by the relative importance of these repair pathways in different cell types. ES cells have relatively high HR activity, compared with other cell types, so that where damage can be repaired by either pathway, loss of Xrcc2 will compromise survival more in ES cells than in MEFs. A similar conclusion was recently reached in studies of Rad54 knockouts, where Rad54-/- ES cells and embryos were hypersensitive to {gamma}-rays, whereas adult Rad54-/- mice were not (39) . Again, sensitivity of the different Rad54-/- cell types to MMC damage did not vary, and in this case, it was argued that adult cells make much greater use of NHEJ than the HR pathway.

Genetic Instability in Xrcc2-Knockout Cells: Comparison to Loss of NHEJ Capacity.
The overall increase in frequencies of aberrations in Xrcc2-/- cells compared with wild-type cells shows that HR repair strongly protects against genetic instability. In primary MEFs, specific aberrations such as complex rearrangements, detached centromeres, and homologue exchanges were seen only in the Xrcc2-/- genotype (Tables 1Citation and 2Citation ). In wild-type mammalian cells, the repair of DSB by HR uses mainly the sister chromatid as a template (40) . The increase in complex rearrangements found in Xrcc2-/- cells shows that some of this DNA damage is channeled into interchromosomal interactions upon loss of HR. It is possible that the loss of Xrcc2 may result in a shift in the balance of DSB repair by recombination from use of sequences on the sister chromatid to those on homologous chromosomes. This idea is potentially supported by the finding of an elevated frequency of homologue:homologue exchanges, compared with that calculated to occur by random interactions. The elevated frequency of detached centromeres in Xrcc2-/- cells is intriguing: sequence duplications between chromosomes appear to be common in pericentromeric regions, and these sequences have been implicated in recurrent chromosomal rearrangements in humans (41) . In this case, HR deficiency may possibly lead to failed interchromosomal exchanges between these sequences, resulting in centromere detachment; alternatively, given the late replicating nature of these regions (42) , HR-deficient cells may be unable to resolve stalled replication forks before the cell enters mitosis, leading to breakage.

DSB may also be repaired by nonconservative mechanisms such as NHEJ or single-strand annealing to yield chromosomal rearrangements (43, 44, 45) . However, recent data show that NHEJ-deficient mouse fibroblasts also have significantly elevated frequencies of chromosomal rearrangements (9, 10, 11, 12, 13) , showing that this pathway is also involved in protecting against such events. Comparison of the NHEJ data sets to our data highlights the fact that the absence of either pathway does not lead to identical responses, strongly suggesting that HR and NHEJ do not successfully back each other up in protecting against instability. For example, a study of chromosomal instability in NHEJ-deficient primary MEFs, also using SKY technology (11) , showed a ratio of rearrangements to fragments of ~1:4 (3:13 scored), whereas this ratio is reversed in Xrcc2-deficient MEFs (36:10 scored; Table 2Citation ). The predominance of rearrangements (translocations, insertions) in the Xrcc2-deficient cells may relate to the damage context: NHEJ is vital in rejoining DNA breaks in G1 cells, whereas HR is important in S phase and G2. In Xrcc2-deficient cells, alteration in damage repair processing in replicating DNA may give greater opportunity for rearrangements either through the enhancement of crossing over relative to gene conversion (see above) or because single-strand annealing is promoted in the absence of gene conversion (16) .

An equally important feature of Xrcc2 deficiency is the extremely high frequencies of chromosome gain and loss (aneuploidy) seen in early passage Xrcc2-/- MEFs (Table 2Citation ; Fig. 4, C and DCitation ). Although MEFs tend to become tetraploid with time in culture, the increased frequency of aneuploidy in the Xrcc2-/- MEFs was clearly in excess of that in wild-type or heterozygous cells (Fig. 1BCitation and Table 2Citation ). We have previously linked aneuploidy in Xrcc2-deficient hamster cells to chromosome missegregation and shown that there is a concomitant occurrence of centrosome defects (30) . We (30) and others (12) have shown that such centrosome defects are not found in NHEJ-deficient cells. Although we have not formally linked centrosome defects to aneuploidy, it is significant that we again see centrosome fragmentation at high frequency in the Xrcc2-/- MEFs. It has been shown recently that unresolved DNA damage or incomplete DNA replication in Drosophila embryos leads to the relocalization of checkpoint kinase 2 to the centrosome, triggering centrosome disruption and mitotic catastrophe (46) . If this mechanism operates in mammalian cells, our data suggest that excess damage resulting from loss of HR repair is effective in triggering the checkpoint kinase 2 pathway, whereas the DSB that are acted on by NHEJ are ineffective. Again, this may relate to the damage context for HR repair, especially the persistence of complex lesions such as stalled replication intermediates in HR-deficient cells (47) .

Haploinsufficiency and Relevance to Carcinogenesis.
We also provide evidence of haploinsufficiency for Xrcc2. This is seen most strongly in the chromosomal aberration frequencies in immortal Xrcc2+/- cells (Table 1)Citation and the increased frequency of centrosome fragmentation (Fig. 5)Citation . This level of instability correlated to a modestly reduced frequency of break-induced HR (Fig. 3A)Citation . Xrcc2 gene expression levels are very low (Ref. 48 and unpublished data), thereby increasing the potential for a reduction in gene copy number to decrease protection against genetic change. Haploinsufficiency in damage-response genes such as Xrcc2 may be of considerable importance: even a relatively weak effect on genetic instability may confer selective advantage, leading to clonal expansion and tumor promotion (49) . This has been seen for example in recent studies of a mouse model for the Bloom’s syndrome (Blm) gene product, involved in recombination, where a similar subtle effect of heterozygosity on genetic instability in cells was shown to lead to enhanced tumor formation in response to challenge with leukemia virus or in an Apcmin/+ genetic background (50) .

The occurrence of genetic instability and, in particular, of aneuploidy and chromosome rearrangement, has been strongly linked to cancer induction (51 , 52) . The link of HR to cancer induction has particularly been made through the discovery of the functional roles of the BRCA genes (see "Introduction"), and in this context, we (53) and others (54) have recently reported that human XRCC2 polymorphism has a marginally significant association with breast cancer incidence. The striking findings of high levels of aneuploidy and chromosome rearrangements, as well as the potential for haploinsufficiency in Xrcc2-deficient cells, reinforce the need to assess further the involvement of members of the HR pathway in carcinogenesis.


   ACKNOWLEDGMENTS
 
We thank David Papworth for statistical calculations.


   FOOTNOTES
 
Grant support: Medical Research Council Studentship (to P. O.) and the European Commission Grant FIGH-CT1999-00010.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: John Thacker, Medical Research Council, Radiation and Genome Stability Unit, Harwell, Oxfordshire OX11 0RD, United Kingdom. Phone: 01235-834393; Fax: 01235-834776; E-mail: j.thacker{at}har.mrc.ac.uk

3 The abbreviations used are: DSB, double-strand break; HR, homologous recombination; NHEJ, nonhomologous end joining; MEF, mouse embryonic fibroblast; ES, embryonic stem; GFP, green fluorescent protein; FISH, fluorescence in situ hybridization; DAPI, 4',6-diamidino-2-phenylindole; BrdUrd, bromodeoxyuridine; MMC, mitomycin C; SCE, sister-chromatid exchange; SKY, spectral karyotyping. Back

Received 7/29/03. Revised 9/11/03. Accepted 9/15/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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