RAD51 is Involved in Repair of Damage Associated with DNA Replication in Mammalian Cells

doi:10.1016/S0022-2836(03)00313-9

J. Mol. Biol. (2003) 328, 521–535

RAD51 is Involved in Repair of Damage Associated with DNA Replication in Mammalian Cells Cecilia Lundin1, Niklas Schultz1, Catherine Arnaudeau1 Atul Mohindra2, Lasse Tengbjerg Hansen3 and Thomas Helleday1,2* 1

Department of Genetic and Cellular Toxicology, Arrhenius Laboratory, Stockholm University, S-106 91Stockholm Sweden 2

The Institute for Cancer Studies, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK 3

Institute of Molecular Pathology, University of Copenhagen DK-2100Copenhagen Denmark

The RAD51 protein, a eukaryotic homologue of the Escherichia coli RecA protein, plays an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) in mammalian cells. Recent findings suggest that HR may be important in repair following replication arrest in mammalian cells. Here, we have investigated the role of RAD51 in the repair of different types of damage induced during DNA replication with etoposide, hydroxyurea or thymidine. We show that etoposide induces DSBs at newly replicated DNA more frequently than g-rays, and that these DSBs are different from those induced by hydroxyurea. No DSB was found following treatment with thymidine. Although these compounds appear to induce different DNA lesions during DNA replication, we show that a cell line overexpressing RAD51 is resistant to all of them, indicating that RAD51 is involved in repair of a wide range of DNA lesions during DNA replication. We observe fewer etoposide-induced DSBs in RAD51-overexpressing cells and that HR repair of etoposide-induced DSBs is faster. Finally, we show that induced long-tract HR in the hprt gene is suppressed in RAD51-overexpressing cells, although global HR appears not to be suppressed. This suggests that overexpression of RAD51 prevents long-tract HR occurring during DNA replication. We discuss our results in light of recent models suggested for HR at stalled replication forks. q 2003 Elsevier Science Ltd. All rights reserved

*Corresponding author

Keywords: RAD51; mammalian cells; homologous recombination; stalled DNA replication forks; DNA double-strand breaks

Introduction Genome integrity is maintained by cell cycle checkpoints in close cooperation with several DNA repair pathways. Any failure in these pathways that lead to unrepaired DNA lesions can contribute to the development of cancer.1 A DNA Present address: C. Arnaudeau, Institute for Cancer Research, 94801 Villejuif, France. Abbreviations used: DMSO, dimethylsulfoxide; DSBs, DNA double-strand breaks; FITC, fluorescein isothiocyanate; HAsT, hypoxanthine-L -azaserinethymidine; hprt, hypoxanthine-guanine phosphoribosyltransferase gene; HR, homologous recombination; NHEJ, non-homologous end-joining; HU, hydroxyurea; PBS, phosphate-buffered saline; PFGE, pulsed-field gel electrophoresis; TdR, thymidine; VP16, etoposide. E-mail address of the corresponding author: [email protected]

double-strand break (DSB) is a potential lethal lesion and is repaired by homologous recombination (HR) or non-homologous end joining (NHEJ).2 The key protein in HR is RAD51, the eukaryotic homologue to RecA in Escherichia coli. The RAD51 protein forms, like RecA, a nucleoprotein filament on single-stranded DNA regions and catalyses (together with other proteins in the Rad52 epistasis group) the search for homologous sequences, strand paring and strand exchange.3,4 RAD51 knockout mice are not viable.5,6 However, in a promotor-controlled RAD51 2/2 DT40 cell line, it has been shown that the knockout phenotype accumulates chromosome breaks during replication and that these cells arrest in the G2/Mphase before entering apoptosis.7 These results suggest an important role for RAD51 during DNA replication; i.e. to maintain genomic stability. Given that HR is important for repair of lesions occurring during DNA replication in both bacteria

0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved

522 and mammalian cells,8 – 10 it is likely that loss of HR repair in RAD51 2/2 cells causes breaks and eventually death of these cells. The activity and level of the RAD51 protein is regulated by p53,11 – 12 which is involved in cellcycle checkpoints. The p53 status appears to affect the rates of RAD51-dependent HR,13 – 15 which might be a direct effect from an interaction between the proteins16 or through the transactivation domain on p53.17 Other factors, such as TGFbeta1, affect the intracellular level of RAD51 and DNA repair, suggesting that the level of RAD51 is biologically important in regulating DNA repair.18 RAD51 interacts with the tumour suppressor BRCA219 and possibly with BRCA1.20 The BRCA2 interaction appears to be important for the RAD51 function, as BRCA22/2 and RAD512/2 cells exhibit similar phenotypes. Both knockouts are embryonic lethal in mice, which in both cases is partially suppressed by additional knockout of p53.5,21,22 Also, both knockout cells accumulate chromosome damage following the S-phase of the cell cycle.7,23 BRCA2 interacts with RAD51 through its BRC repeats encoded by exon 1124,25 and these BRC produce a dominant negative effect on RAD51 function both in vivo and in vitro.24,26,27 However, more important is the fact that the nuclear localization of the RAD51 protein is dependent on BRCA2,24,28 which could be a reason for BRCA22/2 cells to be impaired in the conservative HR pathway involving RAD51.29,30 Given that the BRCA1 2/þ and BRCA2 2/þ genotypes predispose to cancer, HR and RAD51 function might be important in familial predisposition for breast and ovarian cancers.31 This is supported by the fact that RAD51 is altered in certain patients who develop bilateral breast cancer32 and that loss of heterozygocity in the RAD51, RAD52, RAD54, BRCA1 and BRCA2 loci might be related to the development of breast cancer.33 In contrast, overexpression of RAD51 has been associated with human pancreatic adenocarcinoma.34 Such studies suggest that deregulation of HR, resulting in either a decreased or increased frequency might be involved in the carcinogenic process. Thus, it appears that the level and localization of the RAD51 protein is important for HR in mammalian cells. Indeed, previous studies have shown that overexpression of RAD51 increases the spontaneous level of HR,35 – 40 which increases resistance to ionizing radiation.35,37 Increasing evidence from work in bacteria suggests that replication and HR are tightly linked,8 which appears to be the case also in mammalian cells.9,10 Given that RAD51 seems to be important for repair during DNA replication,7 we investigated which substrates, occurring during DNA replication, might trigger HR repair involving RAD51. Since the RAD51 knockout is lethal, we used an established cell line, S8R51.2, which overexpresses RAD51.36 An advantage of using this cell line is that it also carries a 5 kb tandem duplication in the hprt (hypoxanthine-guanine

RAD51 in Repair of Damage during DNA Replication

phosphoribosyltransferase) gene that can be used to assay for HR.41 To induce various forms of replication-associated damage, we used hydroxyurea, thymidine and etoposide (VP16). Hydroxurea and thymidine are used to synchronise growing cells in the S phase of the cell-cycle by two different mechanisms. Hydroxyurea quenches the tyrosyl free radical in the active site of the M2 subunit of ribonucleotide reductase, depleting cells of several deoxyribonucleoside triphosphates, thus stopping replication completely.42,43 This replication block triggers formation of DSBs at or close to replication forks, which are substrates for both HR and NHEJ.10 Thymidine depletes cells of only dCTP and, as a result, slows the progression of the replication fork substantially, but does not stop incorporation of nucleotides into DNA completely.44 No DSB seems to be formed at these slowed replication forks, although a substrate for HR is formed.10 Finally, we used the anti-cancer drug VP16 as a DSB-inducing agent. It induces DSBs by inhibiting the resealing activity of topoisomerase II and has its maximal cytotoxic effect during the S-phase.45 However, it is not clear whether the DSBs formed following treatment with VP16 are induced at replication forks. Here, we show that VP16 induces DSBs at newly replicated DNA more frequently than g-rays and that they differ from DSBs induced by hydroxyurea. Furthermore, we report that RAD51 appears to be involved in the repair of the various types of DNA lesions formed following treatment with hydroxyurea, thymidine or VP16. Finally, we show that induced long-tract HR in the hprt gene is suppressed in RAD51-overexpressing cells, although global HR (occurring in genome overall) may not be suppressed. This suggests that RAD51 is involved in at least two different pathways for HR occurring during DNA replication.

Results Etoposide induces DSBs close to newly replicated DNA that are subsequently repaired by homologous recombination We have shown that hydroxyurea induces DSBs close to or at replication forks.10 To test whether VP16-induced DSBs are formed during DNA replication, we pulse-labelled newly replicated DNA in SPD8 cells for 30 minutes with [14C]thymidine ([14C]TdR), while total cellular DNA in parallel cultures was labelled similarly but for 24 hours. Both cultures were subsequently treated with 5 mM VP16 or 3 mM hydroxyurea for 24 hours or exposed to ionizing radiation (50 Gy). It is worth noticing that the survival following treatment with hydroxyurea is at least 1000-fold higher than for the treatment with ionising radiation.10 Hydroxyurea or ionizing radiation was used as a reference, since their ability to induce DSBs in newly

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Figure 1. VP16-induced DSBs are formed in newly replicated DNA. The DNA of SPD8 cells was labelled with [14C]thymidine ([14C]TdR), either homogeneously for 24 hours or specifically at sites of replication for 30 minutes, prior to exposure to hydroxyurea (HU), VP16 or g-rays. DNA was separated utilising PFGE and visualized by (a) ethidium bromide staining and (b) autoradiography. The size distributions of DNA fragments released from SPD8 cells labelled for 24 hours, i.e. (c) total cellular DNA or 30 minutes, i.e. (d) newly replicated DNA with [14C]TdR and subsequently treated with hydroxyurea (3 mM, blue line), VP16 (5 mM, green line) or g-rays (50 Gy, red line) were determined. It was shown that 22(^5)% and 47% of total [14C]TdR labelled DNA from 24 hours and 30 minutes labelled cells, respectively, were released from the inserts by treatment with hydroxyurea. The corresponding values for VP16 treatments were 36% (24 hours) and 38(^ 1)% (30 minutes) and for g-ray treatments, 60(^ 5)% (24 hours) and 19(^10)% (30 minutes). DNA was separated utilizing PFGE and visualised by autoradiography. DNA fragments were quantified employing Image Gauge software.

replicated DNA has been established.10 Analysis of DNA from these cells by pulsed-field gel electrophoresis (PFGE) revealed that the DNA fragments released by VP16-induced DSBs contained a high proportion of newly replicated DNA (Figure 1(b)), as was the case for hydroxyurea. As expected, the DNA fragments released following g-irradiation contained only a very small proportion of newly replicated DNA, consistent with the random induction of DSBs by this agent occurring mostly between replicons.46 Although both VP16 and hydroxyurea induce DSBs close to newly replicated DNA, the size distribution of the DNA fragments released following the treatments are markedly different. In the case of VP16, the majority of released DNA fragments were large (1.6 Mbp or more; Figure 1(d)). In con-

trast, treatment with hydroxyurea releases a large proportion of DNA fragments that range from 0.2 to 0.6 Mbp in size (Figure 1(d)), which corresponds to the size of a single replicon or a few contiguous replicons.47 Although VP16 is known to induce DSBs, the involvement of HR in the repair of these DSBs has not been investigated thoroughly. To determine if these DSBs are substrates for HR repair, we investigated cell survival following treatment with VP16 in a wild-type (AA8) and an HR-deficient Chinese hamster cell line (irs1SF). We found that the HRdeficient cell line irs1SF (XRCC3 deficient) is hypersensitive to the killing effect of VP16 (Figure 2), in agreement with the results of previous studies.48 – 50 To ensure that the differences seen between these cell lines are due to deficiency in

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RAD51 in Repair of Damage during DNA Replication

HR, we used an irs1SF cell line that has been complemented with a functional XRCC3 gene on a cosmid vector (CXR3).51 We found that the VP16 hypersensitivity is reverted in CXR3 cells (Figure 2), which indicates that HR is required to repair a toxic lesion induced by treatment with VP16. RAD51-overexpressing cells are resistant to etoposide or replication inhibitors

Figure 2. Homologous recombination is required for survival to VP16 damage. Colony outgrowth of the Chinese hamster cell lines AA8 (wild-type), irs1SF (deficient in HR69,70) and CXR3 (irs1SF complemented with XRCC349) upon continuous exposure to VP16. The means (symbols) and standard deviations (bars) from at least four independent experiments are shown.

To investigate if the level of RAD51 is a limiting factor in the HR repair of VP16 damage, we investigated the effects of VP16 in a previously established Chinese hamster cell line, S8R51.2 that has a stably integrated plasmid overexpressing CgRAD51.36 In S8R51.2 cells, the RAD51 protein is overexpressed about two fold (Figure 3(a)), which gives a twofold increase in levels of spontaneous HR.36 We found that the S8R51.2 cell line is more resistant (statistically significant, p , 0.05) to the cytotoxic effects of VP16 than the SPD8 cell line (Figure 3(b)). This is in agreement with a recent study that shows that VP16 resistance in small lung cancer cells is related to the protein level of RAD51, and that overexpression or suppression of the protein level affects the VP16 sensitivity (L.T.H. et al., unpublished results). HR is employed in restoring arrested replication forks in mammalian cells.10 To investigate whether the level of RAD51 determines the rate of restoration of arrested replication forks by HR, we treated S8R51.2 and SPD8 cells with hydroxyurea or thymidine. The S8R51.2 cell line was more resistant than the SPD8 cell line to the cytotoxic effects of either hydroxyurea or thymidine (Figure 4), which indicates that the level of RAD51 is important for efficient restoration of replication forks by HR. RAD51 in repair of etoposide-induced DSBs and apoptosis

Figure 3. (a) RAD51 and actin protein levels in SPD8 and S8R51.2 cells visualized by Western blot. (b) Overexpression of RAD51 increases the survival to VP16. Colony-forming ability in SPD8 and S8R51.2 cells after 24 hours treatment with VP16. The means (symbols) and standard deviations (error bars) from four independent experiments are shown.

To test if increased resistance to VP16 reflects a faster repair of DSBs in S8R51.2 cells, we compared the DSB-repair in this cell line to that in wild-type SPD8 cells (Figure 5(a)). Using PFGE, we observed fewer VP16-induced (2 mM) DSBs in S8R51.2 than in SPD8 cells ( p , 0.05; Figure 5(b)). To determine if RAD51 overexpression reduces those DSBs that occur close to newly replicated DNA, SPD8 and S8R51.2 cells were pulse-labelled with [14C]TdR for 30 minutes and subsequently treated with either 3 mM hydroxyurea or 5 mM VP16. We observed that treatment with VP16 released fewer DNA fragments containing newly replicated DNA in S8R51.2 cells ( p , 0.05; Figure 5(c)). Fewer DNA fragments containing newly replicated DNA were found in S8R51.2 cells following treatment with hydroxyurea (Figure 5(C)). However, this effect was not statistically significant in repeated experiments. The kinetics of the fast repair of DSBs, occurring in the first few hours and thought to involve NHEJ,52,53 was found to be the same in the S8R51.2 as compared to the SPD8 cell line. However, after

RAD51 in Repair of Damage during DNA Replication

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Figure 4. Increased survival to replication inhibitors in cells overexpressing RAD51. Colony outgrowth in SPD8 and S8R51.2 cells following treatment with (a) thymidine or (b) hydroxyurea. The means (symbols) and standard deviations (error bars) from three independent experiments are shown.

24 hours of repair, virtually all DSBs were repaired in the S8R51.2 cell line, while 22% of the induced DSBs remained in the SPD8 cell line (Figure 5(d)). This indicates that the slow repair, generally thought to involve HR,9,10 is quicker in the S8R51.2 cell line, consistent with an increased rate of HR in this cell line.36 Previously, it has been reported that thymidine, although very recombinogenic, does not cause detectable DNA fragmentation in PFGE.10 In agree-

ment with these results, we found that treatment with thymidine at concentrations of up to 20 mM did not induce DNA fragmentation in either the SPD8 or S8R51.2 cell lines (data not shown). Overexpression of RAD51 has been reported to trigger apoptosis54 and to protect against apoptosis induced by g-rays,55 VP16 or UV.56 To measure apoptosis, we used the annexin V assay, which detects translocation of the membrane phospholipid phosphatidylserine to the

Figure 5. DSB repair in SPD8 and S8R51.2 cells after treatment with VP16. (a) DNA fragments (.1.5 Mbp) released by 24 hours treatment with VP16 (2 mM) in SPD8 cells (lanes 1 – 7) and S8R51.2 cells (lanes 8 – 14) after different times of repair. (b) Induction of DSBs in SPD8 and S8R51.2 cells after 24 hours of treatment with 2 mM VP16. The means (columns) and standard deviations (bars) of three experiments are shown. One star denotes a statistical significance of p , 0.05 in Student’s t-test. (c) Hydroxyurea-induced or VP16- induced DSBs at newly replicated DNA in SPD8 or S8R51.2 cells. DNA of SPD8 or S8R51.2 cells was labelled with [14C]thymidine ([14C]TdR) for 30 minutes, prior to exposure to hydroxyurea (HU) or VP16. DNA was separated utilizing PFGE and visualised by autoradiography. DNA fragments were quantified employing Image Gauge software. (d) The amount of DSBs left in SPD8 and S8R51.2 cells after different times of repair (100% represents the amount of DSBs at 0 hours of repair). The means (symbols) and standard deviations (bars) of three experiments are shown.

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Figure 6. Higher level of apoptosis in RAD51-overexpressing cells. (a) The fraction of SPD8 or S8R51.2 cells staining positive with FITCconjugated annexin-V and negative with propidium iodine. The means of apoptotic cells (columns) and standard deviations (error bars) of five experiments are shown. (b) Time of onset of apoptosis in SPD8 and S8R51.2 cells after 24 hours treatment with 2 mM VP16. Control cells were untreated and apoptosis was assessed by staining with annexin-V.

outer leaflet of the plasma membrane in cells that enter apoptosis.55 We found that overexpression of RAD51 by itself increases the number of cells entering apoptosis (Figure 6(a)), in agreement with Flygare and co-workers.54 To test the possibility that the DNA fragmentation detected by PFGE following treatment with VP16 is generated during an apoptotic response to treatment, we measured the onset of apoptosis at various times following a 24 hours exposure of SPD8 or S8R51.2 cells to 2 mM VP16 (Figure 6). Generally, the annexin V assay is thought to detect an early event in apoptosis,55 while DNA fragmentation occurs as a late event.57 Although a small fraction of annexin V positive cells were induced directly following treatment with VP16, the major apoptotic response did not occur in these cells until 12 – 48 hours following treatment with VP16

(Figure 6(b)). Thus, the DNA fragmentation seen directly following 24 hours treatment of SPD8 or S8R51.2 cells with VP16 in the PFGE (Figure 5) is unlikely to be the result of the induction of apoptosis. This is supported by the fact that VP16-induced DSBs are repaired (Figure 5(d)). Furthermore, we found that treatment with 2 mM VP16 induces as much apoptosis in SPD8 as in S8R51.2 cells (Figure 6(b)). This was unexpected, since S8R51.2 cells are less sensitive to VP16 and overexpressed RAD51 has been reported to protect from VP16-induced apoptosis.32 Elevated levels of RAD51 foci in cells overexpressing RAD51 We wanted to investigate if the reported increased level of HR in S8R51.2 cells36 is coupled

Figure 7. Increase in RAD51 foci formation in cells overexpressing RAD51. Visualization of RAD51 foci (yellow) and DNA (red) in SPD8 (a) or S8R51.2 (b) cells. (c) The mean percentages of cells containing spontaneous RAD51 foci (columns) and standard deviations (error bars) from three independent experiments are depicted. (d) The mean recombination frequencies in the hprt gene (columns) and standard deviations (error bars) from six or ten independent experiments are depicted. Two or three stars designate a statistical significance of p , 0.01 or p , 0.001 in Student’s t-test, respectively.

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Figure 8. An illustration of HR in the SPD8 cell line leading to restoration of a functional hprt gene. (a) The hprt gene in the SPD8 cell line contains a duplication of exon 7 and the 30 portion of exon 6. A double line designates the duplication and a single line the original region that was duplicated in connection with the spontaneous mutation. The corresponding mRNA contains only an extra exon 7, since the duplicated exon 6 lacks a 50 splice site and gives rise to a non-functional protein containing 180 amino acid residues. (b) Theoretically, HR employing unequal sister chromatid exchange, intrachromatid exchange, replication fork-associated recombination, single-strand annealing or gene conversion can lead to restoration of the wild-type hprt gene encoding a functional HGPRT protein (c). All pathways for reversion to a functional HPRTþ phenotype is supposed to involve RAD51 except single-strand annealing, which is a non-conservative recombination pathway. Induced recombination frequency in the hprt gene of SPD8 or S8R51.2 cells following exposure to (d) VP16, (e) thymidine or (f) hydroxyurea. The means (symbols) and standard deviations (error bars) from three to six independent experiments are shown.

with an increase in RAD51 foci formation. Not surprisingly, overexpression of RAD51 is coupled with an increase in RAD51 foci formation (Figure 7(c)) as well as the reported increase in spontaneous HR in the hprt gene (Figure 7(d)).36 This is in agreement with a previous study showing that RAD51

foci were three times more abundant when overexpressing RAD51 twofold.56 In the same study, it was reported that RAD51 overexpression caused formation of higher-order nuclear foci; however, we found no such structure in the S8R51.2 cell line (Figure 7(b)).

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RAD51 in Repair of Damage during DNA Replication

Figure 9. (a) Induced RAD51 foci and (b) clonogenic survival in SPD8 or S8R51.2 cells following 24 hours treatment with VP16, thymidine (TdR) or hydroxyurea (HU). The means (columns) and standard errors (error bars) from two to four experiments are shown.

Overexpression of RAD51 reduces long-tract homologous recombination induced during DNA replication Our results show that a high level of RAD51 in S8R51.2 cells is coupled with an increased resistance to VP16, hydroxyurea or thymidine. It has been shown that HR is induced in SPD8 cells following treatments with thymidine,10 hydroxyurea or VP16.58 We wanted to investigate how induced HR is influenced by overexpression of RAD51. HR is investigated in SPD8 and S8R51.2 cells as the frequency of HR between two tandem repeated sequences (5 kb) within the non-functional hprt gene (Figure 8(a)) giving a functional gene that can be selected for with HAsT (hypoxanthine/L azaserine/thymidine) medium (Figure 8(c)). Thus, the reversion frequency in SPD8 and S8R51.2 cells to a functional hprt gene represents the level of HR involving a tract length of at least 5 kb within these cells (Figure 8(b)).36,41 Induction of this longtract HR event was studied after treatment with VP16 in SPD8 and S8R51.2 cells. VP16 induces long-tract HR in a dose-dependent manner in SPD8 and in S8R51.2 cells. However, we observed that considerably less long-tract HR was induced in S8R51.2 cells (Figure 8(d)). The induced levels of HR in both SPD8 and S8R51.2 cells were saturated at higher doses following treatment with thymidine or hydroxyurea. As in the case for VP16, less long-tract HR was induced in the S8R51.2 cell line following treatment with thymidine or hydroxyurea (Figure 8(e) and (f)). From these data, we could not conclude whether the lower levels of induced HR seen in S8R51.2

cells are due to reduction of global HR or just reduction of the long-tract HR event assayed in the hprt gene. In an unconfirmed method to test this, we measured the levels of RAD51 foci forming in SPD8 and S8R51.2 cells following treatment with VP16, thymidine or hydroxyurea. We found that S8R51.2 cells contained more or similar amount of RAD51 positive cells as the wild-type SPD8 cell line (Figure 9(a)). This result, together with increased spontaneous level of HR and less sensitivity in S8R51.2 cells, might indicate that the global rate of HR is not decreased in S8R51.2 cells following treatment with VP16, thymidine or hydroxyurea. A large portion of both SPD8 and S8R51.2 cells form RAD51 foci in response to thymidine or hydroxyurea as compared to VP16-treated cells (Figure 9(a)). To test if this could be explained by an increased amount of toxic lesions present in thymidine or hydroxyurea-treated cells, we investigated the clonogenic survival at these doses (Figure 9(b)). We found that these doses of thymidine or hydroxyurea were less toxic than the VP16 dose given, indicating that repair pathways other than HR participate in repair of VP16-induced damage.

Discussion Although the RAD51 protein plays a critical role in HR, its in vivo function is not fully understood. One reason for this is that the RAD51 knockout phenotype is not viable.5,6 As an alternative

RAD51 in Repair of Damage during DNA Replication

approach, several reports have shown that HR is affected by overexpression of the RAD51 gene.35 – 40 Here, we used the S8R51.2 cell line, which stably overexpresses the CgRAD51 protein about twofold (Figure 3(a)).36 We show that this cell line exhibits the same phenotype as has been reported for cells that overexpress RAD51, i.e. increased levels of RAD51 foci (Figure 7(c)), higher levels of spontaneous HR (Figure 7(d)) and apoptosis (Figure 6). Overexpression of RAD51 has, in one study, been coupled with slower growth and formation of higher-order nuclear RAD51 foci in non-growing primary human PPL cells that are related to p21.56 In contrast, no higher-order nuclear RAD51 foci were detected in the S8R51.2 cells used in the present study. The differences between the study by Raderschall and co-workers56 and ours is likely to be that the S8R51.2 cell line used here is deficient in p53,36 which is known to control p21 expression.59 We used VP16 as a model compound to induce lesions that are repaired by HR. Here, we show that the hypersensitivity to VP16 is restored to wild-type level in the XRCC3 complemented cell line CXR3 (Figure 2), demonstrating that HR is involved in the repair of damage induced by VP16, in agreement with previous reports.46 – 48 Furthermore, we show that the S8R51.2 cell line, which overexpresses RAD51, is more resistant to VP16 (Figure 3(b)). This indicates that RAD51 is rate-limiting in the repair of VP16-induced damage. We suggest that RAD51 is involved in HR repair of VP16-induced DSBs, since virtually all DSBs are repaired in S8R51.2 cells 24 hours after treatment, while 22% of the DSBs remain in SPD8 cells (Figure 5(d)). This is in agreement with the notion that HR repair of DSBs is slow.9,10 Also, it appears likely that the rate of NHEJ is independent of the level of RAD51, since the fast repair of VP16-induced DSBs is similar between SPD8 and S8R51.2 cells. VP16, hydroxyurea and thymidine are all potent inducers of HR (Figure 8). This implies that one or more recombinogenic lesions are formed following treatment with these compounds. To determine whether these compounds form a uniform recombination lesion, we investigated the characteristics of the released DNA fragments using PFGE. gIrradiation (50 Gy) released only 19(^ 10)% of the pulse-labelled DNA from the plug and 60(^ 5)% of total DNA. This low level of release following g-irradiation may be explained by the specific incorporation of a high proportion of the [14C]thymidine at active replication forks into replicons that form bubble structures. Such structures will be obstructed in their movement through an agarose gel.60 – 62 Our observation that VP16 is much more efficient than g-rays in the release of DNA labelled at sites of replication indicates that VP16induced DSBs convert replicon bubbles into linear DNA fragments that migrate into agarose gels.60 – 62 This indicates that some VP16-induced DSBs are associated with replicating DNA. Although both hydroxyurea and VP16 induce DSBs close to

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newly replicated DNA, the released DNA fragments differ markedly in size (Figure 1(d)). It has previously been shown that topoisomerase II activity is active behind the replication fork and that this activity is involved in precatenane removal.63 Our data are in agreement with the hypothesis that VP16-induced DSBs occur behind replication forks, since VP16 releases newly replicated DNA fragments as efficiently as other DNA fragments (38(^ 1)% and 36%; see Figure 1). The reason why treatment with VP16 does not release newly replicated DNA fragments as efficiently as hydroxyurea could be that hydroxyurea might induce DSBs specifically at replication forks.10 Although thymidine induces HR very potently, there are no DNA fragments released from cells following treatment with thymidine (data not shown).10 Thus, the recombinogenic lesion formed during DNA replication following treatment with thymidine seems not to include a DSB. In conclusion, our data suggest that VP16, hydroxyurea and thymidine all cause different lesions during DNA replication. We found that the S8R51.2 cell line is less sensitive to VP16, and to hydroxyurea and thymidine (Figure 4). The increased resistance to these agents is likely to reflect the involvement of RAD51 in the repair of cytotoxic DNA lesions formed by these agents. This observed resistance to DNAdamaging agents is in agreement with previous studies showing that RAD51-overexpressing cells are less sensitive to g-rays,35,37 VP16 (L.T.H. et al., unpublished results) or mitomycin C.64 More importantly, since VP16, hydroxyurea and thymidine appear to produce different DNA lesions during DNA replication, our data suggest that RAD51 (and HR) is involved in repair of several kinds of lesions during DNA replication. This indicates that HR and RAD51 have a broad substrate specificity and may repair lesions that occur close to replication forks (that may or may not include a DSB) and those induced at other locations by VP16. We propose that our results can be explained overall in a current model of how recombination works at DNA replication forks (Figure 10),8 which was proposed originally by Higgins and coworkers in 1978.65 This model suggests that obstructions during DNA replication (e.g. loss of nucleotides caused by hydroxyurea or thymidine or aberrant topoisomerase II function caused by VP16) may lead to stalling of the replication fork. The stalled replication fork may reverse to form a four-way DNA junction (a “chicken foot” including a Holliday junction). In bacteria, this process involves PriA, RecG, RecA and RuvABC,8 and is mediated by positive torsional strain at the progressing replication fork.66 The presence of Holliday junctions in the S phase of the cell cycle in yeast supports the idea that these mechanisms might be conserved.67 We have proposed that the DNA doublestranded tail formed at a chicken foot may serve as a substrate for HR that could restore the

Figure 10. A model for the role of RAD51 in repair at stalled replication forks in mammalian cells. (a) Inhibition of the progression of a replication fork may allow annealing of the nascent leading to the lagging strand, resulting in formation of a chicken foot (b). The chicken foot is a four-way DNA junction containing a Holliday Junction (c). We propose that RAD51 is involved to restore the replication fork at the “tail” of the chicken foot (d). This would explain why we observe less induced DSBs in RAD51-overexpressing cells. Furthermore, we suggest that this HR pathway involves recombination tracts less than 5 kb, since induced long-tract HR in the hprt gene, but not global HR, is suppressed in RAD51-overexpressing cells. (e) Resolution of the Holliday junction present at a chicken foot would result in a DSB, which could serve as a substrate for HR ((f) and (g)) and NHEJ (h). Long-tract HR repair of a DSB can occur on the other repeat (f), which results either reversion to a hprt þ gene (i) or not (j). We suggest that RAD51 is involved in the HR repair of DSBs during DNA replication, since the HR repair of VP16-induced DSBs is more efficient in RAD51-overexpressing cells (thick arrows). The blue line represents template DNA and the dotted red line represents nascent DNA. Yellow regions designate 50 repeat in the hprt gene and green regions the 30 repeat.

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RAD51 in Repair of Damage during DNA Replication

replication fork.10 Although this substrate would include a DNA end, it would not include a DSB. We suggest that RAD51 is involved in the HR event occurring at this DNA tail. This hypothesis is supported by our observation that fewer DSBs are found following treatment with VP16 or with hydroxyurea in S8R51.2 cells. We propose that overexpressed RAD51 may rescue stalled replication forks before these are cleaved into DSBs. The idea that RAD51 is involved in HR repair at a chicken foot tail is supported by our observation that RAD51 appears to be involved in the repair of damage following treatment with thymidine (Figure 6(A)). We found that VP16, hydroxyurea and thymidine all induce considerably less long-tract HR in the hprt gene in RAD51-overexpressing cells (Figure 8(d) – (f)). This could be explained by shorter recombination tracts when recombination occurs at a DNA tail of a chicken foot (Figure 10(c) and (d)). Recombination using shorter recombination tracts could be favoured at this tail, since the absence of a DSB keeps sister chromatids close to each other (Figure 10(c) and (d)). However, when the chicken foot is cleaved into a DSB by resolvases, the free DNA end may recombine with either of the duplicated copies in the hprt gene, increasing the possibility of long-tract HR (Figure 10(f)). Furthermore, we suggest that the RAD51 protein is involved in HR repair of DSBs at replication forks (Figure 10(f) and (g)), since HR repair of VP16-induced DSBs seem faster in RAD51-overexpressing cells (Figure 5(d)). Thus, we suggest that RAD51 is involved in two pathways for HR at stalled replication forks (Figure 10). Although induced long-tract HR events in the hprt gene were suppressed severely in RAD51overexpressing cells, the RAD51 foci formation were not suppressed. In fact, the RAD51 foci levels were induced to a higher or similar level in RAD51-overexpressing cells as compared with wild-type cells (Figure 9). The nature of RAD51 foci is unknown and it is uncertain whether these foci represent a quantitative measurement of global HR. However, given the overall increased spontaneous level of HR, increased survival and RAD51 foci following treatments in RAD51-overexpressing cells, we suggest that only long-tract HR and not global HR is suppressed in these cells. Thus, it appears that the efficiency of HR repair is higher when overexpressing RAD51. Given that fewer DSB lesions are induced and that the DSB repair rate and survival to treatment is higher in RAD51-overexpressing cells, it may appear that high levels of RAD51 would be advantageous to cells. However, in this respect, it is worth noticing that the level of spontaneous long-tract HR is increased in RAD51-overexpressing cells. These long-tract recombination events may lead to gene rearrangements, as in the case in the hprt gene. We suggest that the intracellular levels of the RAD51 protein is kept at a fine balance in which the advantages for repair is weighed with the disad-

vantage of having higher levels of gene rearrangements. This could be the reason why the RAD51 protein is kept in the cytosol in cells and translocated into the nucleus only when needed.24,28 Two earlier studies investigated the effect of RAD51 overexpression on DSB-induced HR with contradictory results. Lambert & Lopez report an increase in HR when overexpressing MmRAD51, while Kim and co-workers report a reduction in HR when overexpressing human RAD51, both using the I-Sce I-system in hamster cells.38,39 Our results are in line with those reported by Kim and co-workers.39 The length of the repeated sequences differs between the hprtand the I-Sce I systems. The hprt system reported here having the longest repeats (5 kb) followed by that of Kim and coworkers (1.4 kb) and that of Lambert & Lopez (0.7 kb). Induced HR was suppressed severely in the hprt gene reported here, while the induced HR was suppressed slightly the 1.4 kb construct.39 Induced HR was increased in the 0.7 kb construct,38 which could imply that the tract length may influence HR pathways that involve RAD51. In conclusion, we show that a portion of VP16 induces DSBs are formed close to newly replicated DNA, and that they differ from DSBs induced by hydroxyurea. Furthermore, we report that RAD51 appears to be involved in the repair of various types of DNA lesions formed following treatments with hydroxyurea, thymidine or VP16, suggesting that HR repair has a broad substrate specificity in mammalian cells. Finally, we show that induced long-tract HR in the hprt gene is suppressed in RAD51-overexpressing cells, although the global HR may not be suppressed. We suggest that RAD51 is involved in two pathways for HR occurring during DNA replication, which is in agreement with the current model for recombination at stalled replication forks.

Materials and Methods Cell lines The SPD8 cell line used in this study was isolated originally on the basis of a spontaneous mutation in the hprt gene of V79 Chinese hamster cells. Analysis of its hprt gene by Southern blotting and sequencing revealed a 5 kb tandem duplication including exon 7, intron 6 and the 30 portion of exon 6,41,68 resulting in expression of a non-functional, truncated HGPRT protein. SPD8 was previously shown to arise by non-homologous recombination and to revert spontaneously by homologous recombination.41 The S8R51.2 cell line derives from SPD8 and possesses a stably integrated vector overexpressing CgRAD51 twofold over endogenous expression levels.36 Only those S8R51.2 cells with a low passage number were used in experiments to minimize the risk of loosing RAD51 overexpression due to an increased rate of apoptosis caused by RAD51 overexpression. The SPD8, S8R51.2 cell lines and revertants thereof were cultured at 37 8C in HMEM (minimum essential medium containing Hank’s salts, along with 9% (v/v) fetal calf serum, 1.8 mM L -glutamine and 90 units/ml of

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penicillin/streptomycin). In the case of the SPD8 and S8R51.2 cells, the medium was supplemented with 6thioguanine (5 mg/ml) in order to kill cells that undergo spontaneous reversion. The growth-rate and plating efficiency of the SPD8 and S8R51.2 cell lines are identical.36 The irs1SF and CXR3 cell lines used in this study originate from AA8 Chinese hamster ovary cells. The irs1SF cell line is deficient for the XRCC3 gene,49 which is one out of five Rad51 paralogs. It has been found to be impaired in the Rad51-mediated HR, induced by a single DSB,69,70 resulting in hypersensitivity to X-rays and crosslinking agents.46 – 48 The CXR3 cell line is an irs1SF cell line complemented with hXRCC3 on a cosmid.49 The AA8, irs1SF and CXR3 cell lines were cultured in Dulbecco’s minimum essential medium (DMEM), with the addition of 9% fetal calf serum and 90 units/ml of penicillin/streptomycin at 37 8C and 5% (v/v) CO2 atmosphere.

RAD51 in Repair of Damage during DNA Replication

minutes incubation with [14C]thymidine (14.4 mM, 27.75 kBq/ml) or with 2 £ 106 cells four hours prior to [14C [thymidine labeling (0.48 mM, 0.925 kBq/ml) for 24 hours. Flasks were rinsed twice with 10 ml of Hank’s balanced salt solution (GIBCO) before initiation of 24 hours of treatment with VP16, hydroxyurea or thymidine in HMEM. In the case of treatment with g-rays (137Cs, 10.6 Gy/min), 1 £ 106 cells were melted into each agarose insert and irradiated. After 24 hours of treatment, the cells in the flasks were released by trypsinisation and 1 £ 106 cells melted into each agarose insert, treated and separated by PFGE as described above. After separation, the DNA was transferred from the gel to a nylon membrane according to the manufacturer’s protocol (Hybond-N, Amersham Pharmacia Biotech). The membrane was then dried for two hours at 80 8C and exposed to a phosphoimager plate for 18 hours before quantification employing Image Gauge software (FLA-3000, Fujifilm).

Toxicity assays In the colony outgrowth assay (used for the AA8, irs1SF and CXR3), 500 cells were plated onto a Petri dish (B 100 mm) four hours prior to a continuous treatment with VP16 dissolved in dimethylsulfoxide (DMSO) at a final concentration of 0.2% (v/v). After seven to 12 days, when colonies were observed, the plates were harvested and the colonies were fixed and stained using methylene blue (Merck) in methanol (4 g/ l). Colonies containing more than 50 cells were counted. For the clonogenic survival assays, flasks (75 cm2) were inoculated with 1.5 £ 106 cells each four hours prior to treatment. VP16 dissolved in DMSO (0.2% final concentration) was added and incubation performed for 24 hours. Hydroxyurea and thymidine were dissolved in medium prior to 24 hours of treatment. After treatments, the cells were rinsed three times with 10 ml of Hank’s balanced salt solution (GIBCO) and 30 ml of HMEM added before allowing the cells to recover for 48 hours. After this recovery, the cells were released by trypsinization and counted. The clonogenic survival was determined by seeding two dishes/each group (500 cells/dish) in 10 ml of medium. After seven days of growth, colonies were fixed and stained using methylene blue (4 g/l in methanol). Colonies containing more than 50 cells were counted. Pulsed-field gel electrophoresis In the repair assay, flasks were inoculated with 5 £ 106 SPD8 or S8R51.2 cells for four hours prior to treatment with 2 mM VP16. After 24 hours of treatment, the cells were released from the flask by trypsinisation and 1 £ 106 cells melted into each agarose insert (75 ml, 1% (w/v) InCert Agarose, BMA). Inserts were kept in HMEM at 37 8C for repair. At given time-points, the inserts were transferred to 0.5 M EDTA, 1% N-laurylsarcosyl and proteinase K (1 mg/ml) and incubated at 50 8C for 48 hours and thereafter washed four times in TE buffer prior to loading onto an agarose separation gel (1% (w/v) Chromosomal grade agarose, Bio-Rad). Separation was performed on CHEF DR III equipment (BioRad; 1208 field angle, 60-240 seconds switch time, 4 V/cm) for 24 hours. The gel was stained overnight with ethidium bromide and subsequently analyzed using Image Gauge software (FLA-3000, Fujifilm). In the [14C]thymidine labelling experiments, flasks were inoculated with 2 £ 106 cells 27.5 hours prior to 30

Immunofluorescence SPD8 or S8R51.2 cells were plated onto coverslips and treated with 5 mM thymidine, 0.2 mM hydroxyurea or 0.2 mM VP16 for 24 hours. Following treatment, the medium was removed, the coverslips rinsed once in PBS (at 37 8C) and fixed in 3% (w/v) paraformaldehyde in PBST (PBS containing 0.1% (v/v) Triton X-100 and 0.15% (w/v) bovine serum albumin) for 20 minutes. The coverslips were rinsed twice in PBS-T prior to incubation with an antibody against RAD51 (H92; Santa Cruz) at a concentration of 1:1000 for 16 hours in 4 8C. The coverslips were rinsed four times (15 minutes each time) in PBS-T followed by one hour of incubation at room temperature with a Cy-3-conjugated goat anti-rabbit IgG antibody (Zymed) at a concentration of 1:500 and then rinsed four times (15 minutes each time) in PBS-T. Antibodies were diluted in PBS containing 3% bovine serum albumin. DNA was stained with 1 mg/ml of To Pro (Molecular Probes). Coverslips were mounted with SlowFade Antifade Kit (Molecular Probes). Images were obtained with a Zeiss LSM 510 inverted confocal microscope using planapochromat 63X/NA 1.4 oil immersion objective and excitation wavelengths of 546 nm and 630 nm. Through-focus maximum projection images were acquired from optical sections 0.50 mm apart and with a section thickness of 1.0 mm. The frequencies of cells containing RAD51 foci were determined by counting at least 300 nuclei in each slide in each experiment. Nuclei containing more than ten foci were classified as RAD51 positive.

Western blot analysis Each protein sample (100 mg/ml) was separated on pre-cast gels (8% – 16% Tris-glycine; BioWhittaker Molecular Applications) and transferred to nitrocellulose membrane (Immun-Blot PVDF, BioRad) using a transblot semi-dry transfer unit (BioRad). This membrane was blocked in 5% (v/v) milk for one hour and the Rad51 protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnologies) at a 1:200 dilution in 3% milk overnight. Anti-rabbit peroxidase conjugates (Santa Cruz Biotechnologies) were used as a secondary antibody at a dilution of 1:50,000. Immunoreactive protein was visualised using ECL reagents (Amersham Pharmacia) following the manufacturer’s instructions.

RAD51 in Repair of Damage during DNA Replication

Apoptosis measurements 6

Flasks were inoculated with 1 £ 10 SPD8 or S8R51.2 cells four hours prior to initiation of 24 hours treatment with 2 mM VP16. Following treatment, the medium was removed, the flasks rinsed twice with 10 ml of PBS; 11 ml of medium was then added. At various timepoints, cells were trypsinized and resuspended with medium containing any floating cells from that sample. The cells were pelleted by centrifugation and resuspended for apoptosis analysis with fluorescein isothiocyanate (FITC)-conjugated annexin-V and propidium iodine (PI) (ApoTarget, Biosource International) according to the manufacturer’s protocol. Samples were analysed by flow-cytometry (Becton-Dickenson FACSort, 488 nm laser), and the percentage of apoptotic cells was determined by the fraction of live cells (PI-negative) bound with FITC-conjugated annexin-V. Recombination assay Each flask (75 cm2) was inoculated with 1.5 £ 106 cells four hours prior to treatment. VP16 dissolved in DMSO (0.2% final concentration) was added and incubation was performed for 24 hours. Hydroxyurea and thymidine were dissolved in medium prior to 24 hours of treatment. After treatments, the cells were rinsed three times with 10 ml of Hank’s balanced salt solution (GIBCO) and 30 ml of HMEM was added before allowing the cells to recover for 48 hours. After this recovery period, the cells were released by trypsinization and counted. HPRTþ revertants were selected for by plating three dishes with each cell suspension (3 £ 105 cells/ dish) in the presence of HAsT (50 mM hypoxanthine, 10 mM L-azaserine, 5 mM thymidine). The clonogenic survival was determined by seeding two dishes/each group (500 cells/dish) in 10 ml of medium. After seven days, cells on the cloning plates were fixed with methylene blue (4 g/l in methanol) and the colonies scored. For selection plates, fixation and scoring were performed four days later.

Acknowledgements We thank Dr Larry Thompson for providing the AA8, irs1SF and CXR3 cell lines, Mr Dan Flower for technical assistance, Dr Mark Meuth, Klaus Erixon and Dag Jenssen for valuable discussions, ¨ nfelt for use of the microscope. and Dr Agneta O This investigation was supported by the Swedish Cancer Society, the Lawski Foundation, the Swedish National Board for Laboratory Animals, the Swedish Fund for Research without Animal Experiments and Yorkshire Cancer Research.

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Edited by J. Karn (Received 19 December 2002; received in revised form 26 February 2003; accepted 4 March 2003)