Spontaneous homologous recombination is decreased in Rad51C-deficient hamster cells

DNA Repair 3 (2004) 1335–1343

Spontaneous homologous recombination is decreased in Rad51C-deficient hamster cells Guido A. Drexler a , Sandra Rogge a , Wolfgang Beisker b , Friederike Eckardt-Schupp a , Malgorzata Z. Zdzienicka c,d , Eberhard Fritz a,e,∗ a

Institute of Molecular Radiobiology, GSF-National Research Center for Environment and Health, Neuherberg D-85758, Germany b Flow Cytometry Group, GSF-National Research Center for Environment and Health, Neuherberg D-85758, Germany c Department of Toxicogenetics, Leiden University Medical Center, 2333 AL, Leiden, The Netherlands d Department of Molecular Cell Genetics, The Ludwik Rydygier University of Medical Sciences, 85-094 Bydgoszcz, Poland e Institut für Molekulare Biotechnologie, Jena D-07745, Germany Received 1 October 2003; received in revised form 4 May 2004; accepted 5 May 2004 Available online 7 June 2004

Abstract The Chinese hamster cell mutant, CL-V4B that is mutated in the Rad51 paralog gene, Rad51C (RAD51L2), has been described to exhibit increased sensitivity to DNA cross-linking agents, genomic instability, and an impaired Rad51 foci formation in response to DNA damage. To directly examine an effect of the Rad51C protein on homologous recombination (HR) in mammalian cells, we compared the frequencies and rates of spontaneous HR in CL-V4B cells and in parental wildtype V79B cells, using a recombination reporter plasmid in host cell reactivation assays. Our results demonstrate that HR is reduced but not abolished in the CL-V4B mutant. We thus, provide direct evidence for a role of mammalian Rad51C in HR processes. The reduced HR events described here help to explain the deficient phenotypes observed in Rad51C mutants and support an accessory role of Rad51C in Rad51-mediated recombination. © 2004 Elsevier B.V. All rights reserved. Keywords: Homologous recombination; Rad51 paralogs; DNA damage; Ionizing radiation; Cross-linking agents

1. Introduction Homologous recombination (HR) has emerged as a major pathway for the correct repair of DNA double strand breaks (DSB), DNA interstrand cross-links, and other types of DNA damages in mammalian and yeast cells. In particular, DSBs are produced by ionizing radiation and radiomimetic chemicals, and they likely occur spontaneously during DNA replication [1,2]. When left unrepaired, a single DSB may trigger cell cycle checkpoints and cause cell death [3,4]. The correct repair of DSBs by HR can only occur when extensive regions of DNA homology are available, either shared between the homologous chromosomes in diploid mammalian cells or, more likely, between the replicated sister chromatids ∗ Corresponding author. Tel.: +49 3641 656371; fax: +49 3641 656335. E-mail address: [email protected] (E. Fritz).

1568-7864/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2004.05.002

[5]. In the latter context, the importance of HR processes in repair of stalled, and broken replication forks has recently been described [6–10]. The mammalian Rad51 protein, a functional homolog of bacterial RecA, is the key recombination protein promoting the exchange between homologous DNA sequences [11]. Rad51 forms nucleoprotein filaments on DNA and is redistributed upon DNA damage in nuclear foci on the damaged sites [12,13]. The nuclear foci contain additional proteins involved in HR and are thought to represent the biochemical complexes performing DNA repair by HR on the one hand [14–16], and regulating cellular survival responses on the other hand [17]. Besides Rad51 and the closely related meiosis-specific DMC1 protein, five Rad51 paralogs (XRCC2, XRCC3, Rad51B, Rad51C, Rad51D) exist in mammalian cells which share 20–30% sequence similarity with Rad51 and with each other [18–23]. The paralogs were first implicated in HR based on their sequence similarity to

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Rad51, which is mainly concentrated in the central part of the proteins that include two Walker A and B motifs potentially involved in ATP hydrolysis [24]. A functional role of the Rad51 paralogs in HR was subsequently supported by both phenotypic data obtained in respective vertebrate mutant cells [22,25–27] as well as biochemical interaction and cellular co-localization studies showing physical association between Rad51 paralogs and the Rad51 protein [28–31]. Most notably, a direct role of the XRCC2 and XRCC3 proteins in recombinational DNA repair has been established in respective hamster cell mutants by using chromosomally integrated systems that artificially create site-specific DSBs and detect their repair by HR [32,33]. Rad51C has been studied extensively in chicken DT40 cells where a role in HR has been deduced based on the hampered Rad51 foci formation, increased spontaneous chromosome aberrations, the reduced formation of SCEs, and the reduced efficiency of targeted integration of genomic DNA [27]. The latter two phenotypes have been attributed directly to reduced HR processes. The mammalian Rad51C gene has been found to be inactivated in two different mitomycin C (MMC)-sensitive hamster cell mutants. The irs3 hamster mutant contains a point mutation in exon 6 of the Rad51C gene which ultimately abolishes protein expression [34]. Another hamster cell line, CL-V4B, carries a 132 bp deletion corresponding to exon 5 of the Rad51C gene [35]. Deletion of exon 5 results in loss of the Walker B box that destroys the ATP binding site and the core domain of the Rad51C protein. Functional analyses of both mutant cell lines revealed increased cellular sensitivity to DNA damaging agents, especially cross-linking agents, impaired DNA damage-induced formation of Rad51 nuclear foci, and increased levels of both spontaneous and MMC-induced chromosome aberrations. Most notably, Rad51C-deficient hamster cells show reduced spontaneous and MMC-induced levels of SCEs that may reflect HR processes associated with crossing-over events. In this report, we directly analyzed the impact of mammalian Rad51C protein on HR events, by using a recombination reporter plasmid capable of detecting cellular HR processes that can reconstitute a functional eGFP gene (Drexler et al., this issue). We applied this assay in Rad51C-deficient CL-V4B [35] cells and their parental progenitor V79B cells in order to measure spontaneous chromosomal HR. The measurement of both HR frequencies and HR rates demonstrated clearly reduced HR in Rad51C-deficient cells. Our data confirm a direct role of mammalian Rad51C in HR processes and, thus, support earlier indirect conclusions drawn from DT40 chicken cells lacking Rad51C. The finding of reduced HR in Rad51C-deficient mammalian cells suggests inefficient DNA repair by HR, which in turn provides an explanation for the concomitant cellular deficiencies, like hypersensitivity to DNA damaging agents, increased chromosome aberrations, and reduced SCE levels.

2. Materials and methods 2.1. Cell culture and transfection of pGrec V79B hamster fibroblasts and their Rad51C-deficient CL-V4B derivatives [35] were cultured in Nutrient Mix F-10 with Glutamax (Gibco BRL) and 10% FCS (PAA Laboratories, Linz, Austria). Under these conditions, the generation times of V79B and CL-V4B cells were 11 and 15 h, respectively. The cells were routinely transfected with pGrec, using GenePorter Transfection Reagent (Gene Therapy Systems Inc., San Diego, USA) according to the manufacturer’s protocol. Cells with stable uptake of the vector were selected in hygromycin-containing media (Calbiochem) at 400 U/ml for V79B, and 350 U/ml for CL-V4B cells. Since the EBV-based pGrec vector (Fig. 1) can not autonomously replicate as an episome in rodent cells, stable transfection selects for chromosomal integration of the vector. Characterization of integration events and calculation of the pGrec copy number was performed following genomic Southern hybridizations according to standard procedures. The full length eGFP-ORF was used as a hybridization probe following amplification and simultaneous radioactive labelling (see Drexler et al., this issue). Recombination within pGrec may occur between the two differentially mutated eGFP alleles that share a 383 bp overlapping region (bp 70 to bp 452) of the eGFP-ORF, thus reconstituting a functional, wildtype eGFP allele. Recombination was detected as green, eGFP-specific cellular fluorescence in microscopic or flow cytometric analyses. 2.2. Determination of recombination frequencies In each experiment, V79B and CL-V4B cells were freshly transfected in parallel with the pGrec recombination vector and selected for stable uptake and integration of the vector, using hygromycin. All individual hygr colonies on each plate were scored once for the presence of living eGFP+ cells by UV microscopy. Before microscopy of the cells in 60, 100, or 150 mm dishes, the growth media was removed and replaced by PBS. Microscopic detection and documentation of living eGFP+ cells were performed at 60× magnification, using a BH-2 RCA microscope equipped with an 490 nm UV lamp (Olympus Optical, Hamburg), and a SC36 type 12 camera (Olympus). Colonies containing eGFP+ cells fell into three arbitrary categories, either containing a single eGFP+ cell, a local cluster of multiple eGFP+ cells, or an eGFP+ colony comprised of >50% eGFP+ cells. In very rare cases (3%), a single colony contained two separate eGFP+ events that were scored individually. In control experiments, stable transfections of expression vectors carrying solely one of the mutated eGFP alleles (Drexler et al., this issue) never led to eGFP+ cells in microscopic or flow cytometric analyses. In contrast, the expression vector carrying a wildtype eGFP allele produced >90% cells exhibiting eGFP fluorescence (data not shown). In each experiment,

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Cellular HR events leading to GFP+ cells:

Recombination vector and eGFP alleles:

wt GFP TGAGAGATCTTCA

Intramolecular

GFP-FS 5‘∆GFP

SV40 polyA

RSVLTR

EBNA-1

Gene conversion

HR

SV40GFP-FS puromycin 5‘∆-GFP

TK-polyA

pGrec

Hygromycin

12913 bp

TKPromotor

Amp/ori

Single strand annealing

Intermolecular exchange (unequal sister chromatid exchange)

Fig. 1. Schematic presentation of the pGrec recombination vector and cellular HR events: All episomal vectors contain the wildtype or mutated eGFP alleles cloned behind the constitutive RSV-LTR promotor. In pGrec, the mutated eGFP alleles are cloned head to tail and separated by a puromycin resistence cassette. The negative control vectors harbour the mutated eGFP alleles following deletion (GFPdelta) or insertional inactivation (GFP-FS) of the wildtype allele. The positive control vector contains the wildtype eGFP allele.

either one or multiple parallel dishes were scored microscopically, at defined times post transfection. Since, the CL-V4B cells had a slightly prolonged generation time compared to V79B control cells, they were allowed to grow longer on the dishes before eGFP+ cells were scored. That way, in each experiment the average number of cells per colony was comparable between Rad51C-deficient CL-V4B cells and their V79B controls. The frequency of HR, or the mean frequency from parallel dishes, was calculated from the number of hygr colonies containing eGFP+ events within the total number of colonies analysed. Analyses of eGFP fluorescence at different times following pGrec transfection did not reveal significant changes in recombination frequencies between 9 and 22 days post transfection. The experiments for determining recombination frequencies were performed and scored by two independent individuals. 2.3. Determination of recombination rates in fluctuation experiments For measurement of recombination rates, single clones transfected with pGrec were isolated and expanded in order to determine the integrity and number of recombination cassettes by Southern analyses. Suitable single clones were subjected to fluctuation experiments as described earlier [36], and subsequent detection of recombined, eGFP+ single cells was achieved by flow cytometry. For flow cytometry, living cells were harvested by mild trypsinization, washed two times in cold PBS, and analyzed on a FACStarPLUS platform (Beckton Dickinson, San Jose,

USA). An argon ion laser at a wavelength of 476 nm was used to excite eGFP fluorescence (excitation maximum of the eGFP protein sg25 at 474 nm, [37]), and detection of specific eGFP fluorescence was achieved using a 510 nm bandpass filter with 10 nm bandwidth (emission maximum of the eGFP protein sg25 at 509 nm, [37]). Conformational flow cytometric analyses were performed using a FACS Vantage platform (Beckton Dickinson). Recombination rates were determined, based on the number of eGFP+ events, and the total number of cells analysed. They were calculated as recombination events within pGrec/cell/generation, according to the equations of Luria and Delbrück, using fluctuation tables [38]. Due to the very low rate of spontaneous HR, about 30% of fluctuation experiments (three out of nine experiments for parental cells, and two out of five experiments for Rad51C-deficient cells) did not contain enough cells to detect at least one eGFP+ event. For statistical reasons, such experiments failing to detect eGFP+ cells due to low cell numbers were omitted in the calculation of recombination rates.

3. Results 3.1. Reduced spontaneous recombination frequency in Rad51C-deficient cells A number of indirect evidences from cellular and biochemical studies suggested an involvement of the

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mammalian Rad51C protein in HR processes. We attempted to measure more directly HR events in Rad51C-deficient hamster cells by using the pGrec recombination vector (Drexler et al., this issue). Following stable chromosomal integration of pGrec, the conversion of two differentially mutated and inactivated eGFP alleles to a functional wildtype eGFP allele by HR can be detected based on cellular eGFP fluorescence (Fig. 1). The HR mechanisms detectable by pGrec include gene conversion and intermolecular exchange (unequal SCE), both of which most likely depend on the presence of sister chromatids, as well as intramolecular exchange and single-strand annealing. In nine independent experiments, V79B hamster fibroblasts, and their Rad51C-deficient CL-V4B derivatives, were freshly transfected in parallel with pGrec. hygr colonies, each representing individual genetic pGrec integration events, were allowed to grow during 9–22 days post transfection until they were monitored microscopically for

eGFP-fluorescent cells. In each experiment, the CL-V4B cells were given an extended cultivation time before scoring, according to their prolonged generation time, to ensure that colonies with similar average cell numbers were compared between the Rad51C-decient CL-V4B cells and their progenitor wildtype cells. In Table 1, the combined data gathered from nine experiments are shown. Since all hygr colonies were scored on each dish, the total number of colonies analyzed, divided by the number of dishes, represents the transfection efficiency for each experiment, which was not obviously different between the wildtype and Rad51C-deficient cells. The number of independent colonies carrying eGFP+ events, either fluorescent single cells, cell clusters or fluorescent colonies, within the total number of hygr colonies represents the spontaneous recombination frequency. In each of the nine experiments, the number of eGFP+ events was drastically decreased in Rad51C-deficient cells compared to

Table 1 Reduced recombination frequencies in Rad51C-deficient CL-V4B cells Cell line

Number of dishes analysed (day post transfection)

Total number of colonies analyzed

eGFP+ events Single cells

Sector

Colony

Total

Mean recombination frequency

Experiment 1 V79B CL-V4B

3 (9, 13,14) 4 (15, 19, 20, 21)

715 968

54 0

49 2

7 0

110 2

15.4 0.2

Experiment 2 V79B CL-V4B

1 (12) 1 (20)

212 223

24 7

27 6

4 0

55 13

25.9 5.8

Experiment 3 V79B CL-V4B

1 (10) 1 (14)

379 395

42 4

40 0

1 0

83 4

21.9 1.0

Experiment 4 V79B CL-V4B

3 (14) 3 (15)

643 1057

27 5

40 5

2 0

69 10

10.7 0.9

Experiment 5 V79B CL-V4B

1 (10) 1 (14)

278 392

13 0

10 0

1 0

24 0

8.4 <0.3

Experiment 6 V79B CL-V4B

1 (14) 1 (20)

368 267

10 0

9 0

0 0

19 0

5.2 <0.37

Experiment 7 V79B CL-V4B

2 (10, 14) 2 (12, 17)

638 348

66 4

18 2

3 0

87 6

13.6 1.7

Experiment 8 V79B CL-V4B

1 (14) 3 (20, 21, 22)

368 763

10 12

9 5

0 0

19 17

5.2 2.3

Experiment 9 V79B CL-V4B

2 (9, 10) 2 (14, 15)

565 677

35 0

20 0

1 0

56 0

9.9 <0.14

In each experiment, wildtype V79B and Rad51C-deficient CL-V4B were transfeced in parallel with pGrec and the resulting hygr colonies were monitored microscopically for eGFP expressing cells after growth for 9–22 days. eGFP+ events were scored in three different categories, single cell, sectors and colonies. In very rare cases (<3%), a single colony contained two separate eGFP+ events that were scored individually. The recombination frequency was calculated as the ratio between eGFP+ events and the colony number analyzed. For experiments in which no eGFP+ event was detected in CL-V4B cells (see experiments: 5, 6 and 9), a theoretical recombination frequency was calculated assuming that the next colony analyzed would contain an eGFP+ event. The data of experiments 1–4 and experiments 5–9 were obtained by two independent scorers.

Recombination frequency (%)

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30 V79B CL-V4B

25 20 15 10 5 0 1

2

3

4

5

6

7

8

9

experiment

Fig. 2. Reduced recombination frequency in Rad51C-deficient cells: In 9 independent experiments, V79B wildtype hamster fibroblasts and their Rad51C-deficient derivates CL-V4B were stably transfected with the pGrec recombination vector. After growth for 20 to 35 generations, the resulting hygr colonies were monitored microscopically for eGFP+ events indicating homologous recombination. The frequency of eGFP+ events for each experiment is depicted. Experiments 1–4 and experiments 5–9 were analyzed by 2 independent scorers.

their isogenic control cells. In three experiments, none of the pGrec-containing hygr colonies of the Rad51C-deficient CL-V4B cells produced any eGFP+ cells, in contrast to their wildtype controls (Fig. 2). Within the nine independent experiments, each showing reduced HR frequencies in Rad51C-deficient cells, the number of detected eGFP+ events varied considerably. This effect may occur due to different individual eGFP+ scoring thresholds and may also arise through interexperimental differences of transfection efficiencies that produce an altered distribution of intact pGrec integrations or numbers of integrated pGrec alleles within the hygr colonies. The mean recombination frequencies calculated from all nine independent experiments is 12.91% (±6.7 standard deviation) for wildtype cells and 1.99 (±1.87 standard deviation) for Rad51C-deficient cells. The data clearly demonstrate that, during 9–22 days following stable transfection, Rad51C-deficient cells have a reduced spontaneous chromosomal recombination frequency that reconstitutes a wildtype eGFP allele. 3.2. Reduced spontaneous recombination rates following fluctuation analyses of Rad51C-deficient cells To further confirm reduced HR in Rad51C-deficient cells, we used an alternative approach to measure recombination rates in individual subclones by means of fluctuation analyses. Individual subclones transfected with pGrec were isolated and the integrated pGrec copies were characterized by Southern analyses. Following restriction digestion of the genomic DNA, with EcoRV, hybridization of an eGFP probe detects intact recombination cassettes released from integrated pGrec as a 4 kB band, as exemplified using as control the pGrec plasmid (Fig. 3). Quantitative comparison of the recombination cassettes with the control plasmid suggests that V79B-Grec1, V79B-Grec20 (data

Fig. 3. Southern analysis of integrated pGrec vectors: Genomic DNA isolated from single clones of pGrec-transfected V79B and CL-V4B cells was restriction digested using EcoRV, run on an agarose gel and subjected to Southern blotting and hybridization using a full length eGFP probe. Non-disrupted recombination cassettes of integrated pGrec appear as a 4 kB band, as can be seen in the control samples of digested pGrec plasmid. Based on quantitative comparison to the control pGrec samples, it was calculated that the clones V79B-Grec1, -Grec23 and CL-V4B-Grec6, -Grec11 contain one pGrec copy per cell. The same conclusion was drawn based on hybridisations of these genomic DNA samples following restriction digestion with single cutting enzymes for pGrec, where a single band appears following hybridization with an eGFP probe (data not shown). pGrec, control digest using dilutions of the pGrec plasmid DNA; M, molecular size marker.

not shown), and V79B-Grec23, as well as CL-V4B-Grec6 and CL-V4B-Grec11 contain one integrated pGrec copy per cell. Additional hybridization experiments, following restriction digestion with single cutters for pGrec show only one high molecular band for these cell clones, which further supports a single copy integrated status of pGrec in the respective cells (data not shown). The three wildtype subclones, V79B-Grec1, V79BGrec20, and V79B-Grec23, as well as the two Rad51Cdeficient subclones, CL-V4B-Grec6, and CL-V4B-Grec11, were propagated for fluctuation analyses and subjected to flow cytometric detection of eGFP+ cells. In Table 2, the data obtained from individual fluctuation experiments performed on two different flow cytometers are summarized. pGrec-transfected subclones of the wildtype V79B cell line had recombination rates of 1.22 × 10−6 eGFP+ events/cell/generation for subclone Grec1 (mean value of three experiments), 1.19 × 10−6 for subclone Grec20 (mean value of two experiments), and 0.93 × 10−6 for subclone Grec23 (one experiment). In contrast, Rad51C-deficient derivatives transfected with pGrec showed reduced recombination rates of 0.28 × 10−6 eGFP+ events/cell/generation for subclone Grec6 (mean value of two experiments), and 0.29 × 10−6 for subclone Grec11 (one experiment). When summarized, the mean value from six experiments with three individual pGrec-transfected V79B subclones (mean 1.16, ±0.53 standard deviation) showed an over four-fold increased HR rate as compared to the mean value from three experiments with two individual pGrec-transfected CL-V4B subclones (mean 0.28, ±0.09 standard deviation). Taken together, our analyses of

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Table 2 Reduced recombination rates in Rad51C-deficient cells Cells (subclones)

Control cells V79B-Grec1 V79B-Grec1 V79B-Grec1 (a) V79B-Grec20 (a) V79B-Grec20 V79B-Grec23 (a)

Number of Number of eGFP+ Recombination cells analyzed cells rate (× 10−6 ) (× 106 ) 2.6 2.9 3.0 5.1 2.6 3.1

2 1 11 10 4 3

Mean Rad51C-deficient cells CL-V4B-Grec6 10.4 CL-V4B-Grec6 6.0 CL-V4B-Grec11 6.0 Mean

0.90 0.61 2.16 1.10 1.27 0.93 1.16

1 2 1

0.17 0.39 0.29 0.28

Single clones of V79B wildtype and Rad51C-deficient CL-V4B cells transfected with pGrec were isolated and expanded in order to determine the integrity and number of recombination cassettes. Suitable single clones containing 1–2 integrated copies of pGrec were subjected to multiple independent fluctuation experiments as described earlier [36]. Detection of recombined, eGFP+ single cells was achieved by flow cytometry on a FACStarPLUS platform or on a FACS Vantage (a). Recombination rates were calculated as recombination events within pGrec/cell/generation according to Luria and Delbrück, using fluctuation tables [38].

recombination rates again demonstrate reduced HR events in Rad51C-deficient hamster cells.

4. Discussion We used a host cell reactivation approach to determine frequencies and rates of spontaneous HR within the chromosomally integrated pGrec recombination vector in Rad51C-deficient hamster cells. In nine independent experiments designed to determine HR frequencies, we consistently found significantly decreased numbers of HR events in Rad51C-deficient cells as compared to their isogenic wildtype cells. The mean recombination frequency calculated from all experiments was six-fold reduced in Rad51Cdeficient cells compared to their isogenic control cells. Since our data on HR frequencies rely on the stable transfection of a reporter plasmid, different transfection efficiencies possibly depending on Rad51C function might bias the outcome of our experiments. In fact, in chicken DT40 cells, where targeted integration of genomic sequences into the cellular genome is a rather efficient process, mutations in any of the Rad51 paralogs have been found to reduce targeted integration efficiency, which was interpreted as a reduced capacity to perform HR [27]. However, in our system integration of non-homologous pGrec vector sequences into the mammalian genome is required. In this regard, we found no obvious alterations in stable transfection efficiencies between wildtype and Rad51C-deficient cells in nine independent experiments. Additionally, Southern analyses of

multiple individual transfected subclones did not show obvious differences in the distribution of pGrec copy numbers between wildtype and Rad51C-deficient cells. These data suggest that genomic integration of non-homologous vector sequences into the mammalian genome is not affected by Rad51C deficiency. For our system used to compare HR frequencies between wildtype cells and Rad51C-deficient derivates, we can therefore, rule out an experimental bias of HR detection. We confirmed the finding of reduced spontaneous HR by determining the rates of HR in fluctuation analyses of individual subclones of pGrec-transfected Rad51C-deficient, and control wildtype cells. The subclones used in this assay most likely contain a single pGrec integration, according to Southern analyses. The HR rates measured would thus reflect intrachromosomal recombination events. The mean value from three experiments with two individual pGrec-transfected CL-V4B subclones (mean 0.28, ±0.09 standard deviation) showed an over four-fold decreased HR rate as compared to the mean value from six experiments with three individual pGrec-transfected V79B subclones (mean 1.16, ±0.53 standard deviation). We conclude that reduced HR frequencies, as seen above in the total population of pGrec-transfected CL-V4B colonies, are paralleled by reduced HR rates in individual CL-V4B subclones compared to subclones from wildtype cells. Taken together, Rad51C-deficient cells show reduced spontaneous HR events that correctly restore a functional eGFP allele in the recombination vector. In a recent publication [27], defective recombinational repair of knockout mutants of the Rad51 paralogs in DT40 chicken cells has been described. In this report, Rad51C-deficient DT40 cells, like the other Rad51 paralog mutants, showed approximately three-fold decreased spontaneous and MMC-induced SCE events, which likely reflect HR repair in replicative DNA associated with crossing-over between sister duplexes. In addition, targeted integration of transfected genomic DNA fragments, a mechanism readily detectable in DT40 cells, has been shown to be decreased in Rad51C mutants. Both phenotypes are thought to reflect spontaneous HR events which are suppressed upon mutation of Rad51C or the other Rad51 paralogs, thus strongly supporting our HR data obtained in mammalian cells. However, functional data on HR proteins from chicken DT 40 cells can not be strictly extrapolated to the situation in mammalian cells. In this regard, different functional activities of human Rad51-cDNA in cross-complementing hamster and chicken cells has been observed [27,34]. Also, Rad54 has been found to be required for Rad51 focus formation in mouse [15], but not in chicken DT40 cells [26]. Concerning Rad51C, both the indirect data obtained in chicken DT40 cells as well as our data obtained in mammalian hamster mutants show reduced HR events upon Rad51C deficiency, suggesting a conserved role of this protein in spontaneous HR processes. The MMC-hypersensitive phenotype of mammalian and DT40 cell mutants lacking proteins of the Rad51 family has

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clearly demonstrated that the repair of DNA cross-links, induced by MMC and similar agents, involves HR. While cross-link repair in Escherichia coli and Saccharomyces cerevisiae is performed by the interlinked processes of NER and HR [39,40], its mode of action in vertebrate and mammalian cells is still unclear [41]. The spontaneous HR events detected with our system most likely do not depend on DNA cross-links but rather on HR repair of spontaneous single- or double-strand breaks, e.g., in replicating DNA. These lesions can readily occur in or close to a replication fork, and they have been shown to be repaired by HR mechanisms between sister chromatids, involving Rad51 [10]. In contrast, HR between homologous chromosomes is unlikely to occur at a significant rate in mammalian cells [42]. Rad51C deficiency leads to enhanced spontaneous and DNA damage-induced chromosomal instability in both rodent and DT40 mutants, suggesting that HR is a major pathway for the correct repair of spontaneous and induced DNA damage. For the CL-V4B cells used in our study, an increased level of both spontaneous and MMC-induced chromosome aberrations has been demonstrated. Interestingly, the majority of aberrations in CL-V4B were of the chromatid-type, implying that specifically DNA breaks occurring in S-phase of the cell cycle were processed incorrectly. In addition, a reduced appearance of spontaneous SCEs, and a lack of SCE induction following MMC treatment has been reported for CL-V4B cells. Since, SCEs occur upon DNA damage of replicating chromosomes that is correctly repaired and followed by an crossing-over event, the reduced spontaneous SCE formation, and the lack of SCE induction upon MMC treatment again supports a functional role of mammalian Rad51C on replicating DNA, similar to what was reported for Rad51C-deficient chicken DT40 cells. Based on our experimental set up, we can not rule out the formal possibility that initiation and resolution of HR events may be as common in Rad51C-deficient cells like in wildtype controls, but that many of these were error-prone and, thus, not detectable in our functional assay system. However, the reduced Rad51 foci formation consistently found in Rad51C-deficient cells strongly supports a reduced initiation of HR events, since Rad51-like protein complexes bind preferentially to single-stranded DNA and are expected to recruit Rad51 to sites of damaged DNA. For example, the formation of Rad51 filaments occurs close to the Rad51B-Rad51C-Rad51D-XRCC2 complex [30]. Among the Rad51 paralogs, the Rad51C protein may play a unique role in HR according to its various biochemical functions. Concerning interactions between Rad51 and the paralogs, the Rad51C protein has been demonstrated to be a central player, interacting directly with Rad51, Rad51B, Rad51D, and XRCC3 [29]. Also, Rad51C is present in various multiprotein complexes in human cells [30,31,43–45], implying that it may have multiple roles in HR. In vitro, both Rad51B, and Rad51C have been shown to bind preferentially to damaged DNA and to display ATPase activity in the presence of DNA [46]. Furthermore, only the Rad51C

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protein, however, was found to promote a DNA strand exchange reaction in vitro on oligonucleotide DNA substrates, similar to Rad51, suggesting that Rad51C has a more direct role in HR in addition to its role of recruiting Rad51 to the damaged DNA [46]. Interestingly, Rad51C seems not to be absolutely required for spontaneous HR, since HR events measured as frequencies and rates, are reduced but not completely abolished in the mutant CL-V4B cells. This finding is supported by the approximately three-fold reduced, but not abolished frequency of spontaneous and MMC-induced SCEs found in Rad51C-deficient chicken DT40 cells. The reduced recombination seen in Rad51C-deficient hamster cells is paralleled by HR defects demonstrated in mammalian XRCC2- and XRCC3-deficient mutants [32,33]. Presumably, Rad51 paralogs may have redundant functions and complete Rad51C deficiency is compensated by other Rad51 family members. Alternatively, lack of Rad51C may be bypassed by switching from Rad51 family-dependent HR mechanisms like gene conversion and SCEs to mechanisms that may occur independently from Rad51C, like, e.g., single-strand annealing. The latter is also detectable with our pGrec recombination reporter system. HR defects in mammalian mutant cells have been described for the genomic instability syndromes Ataxia telangiectasia [36,47,48], the murine homolog of Blooms Syndrome [49] and Werner Syndrome [50]. While the former two syndromes show increased cellular homologus recombination, reduced HR has been reported for Werner syndrome cells as well as for BRCA1- and BRCA2-deficient cells [51–53]. In addition, overexpression of Rad51, the key recombinase, has been found in tumors [54]. Taken together, these data strongly suggest that alterations in cellular HR frequencies, either abnormally decreased or increased, may be crucially involved in mutagen sensitivity, genomic instability and tumorigenesis. Acknowledgements We thank Barbara Godthelp, Rebecca E.E. Esvelt-vanLange, Ulrike Hamm, and Klaudia Winkler for technical assistance and Reinier van der Linden for conformational flow cytometric analyses. This work was supported by CEC grant F14PCT950010 (to F. E-S.). Note added in proof Upon completion of this manuscript, reduced homologus recombination has been reported for another Rad51Cdeficient rodent cell line (J Biol Chem. 2003 Nov 14;278(46)45445-50.) References [1] J.E. Haber, DNA recombination: the replication connection, Trends Biochem. Sci. 24 (1999) 271–275.

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[2] S.C. Kowalczykowski, Initiation of genetic recombination and recombination-dependent replication, Trends Biochem. Sci. 25 (2000) 156–165. [3] R.J. Bennett, H.J. Dunderdale, S.C. West, Resolution of Holliday junctions by RuvC resolvase: cleavage specificity and DNA distortion, Cell 74 (1993) 1021–1031. [4] L.C. Huang, K.C. Clarkin, G.M. Wahl, Sensitivity and selectivity of the DNA damage sensor responsible for activating p53-dependent G1 arrest, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 4827–4832. [5] P. Karran, DNA double strand break repair in mammalian cells, Curr. Opin. Genet. Dev. 10 (2000) 144–150. [6] M.M. Cox, M.F. Goodman, K.N. Kreuzer, D.J. Sherratt, S.J. Sandler, K.J. Marians, The importance of repairing stalled replication forks, Nature 404 (2000) 37–41. [7] B. Michel, Replication fork arrest and DNA recombination, Trends Biochem. Sci. 25 (2000) 173–178. [8] C. Arnaudeau, C. Lundin, T. Helleday, DNA double-strand breaks associated with replication forks are predominantly repaired by homologous recombination involving an exchange mechanism in mammalian cells, J. Mol. Biol. 307 (2001) 1235–1245. [9] C. Janz, L. Wiesmuller, Wild-type p53 inhibits replication-associated homologous recombination, Oncogene 21 (2002) 5929–5933. [10] C. Lundin, N. Schultz, C. Arnaudeau, A. Mohindra, L.T. Hansen, T. Helleday, RAD51 is involved in repair of damage associated with DNA replication in mammalian cells, J. Mol. Biol. 328 (2003) 521– 535. [11] P. Baumann, S.C. West, Role of the human RAD51 protein in homologous recombination and double-stranded-break repair, Trends Biochem. Sci. 23 (1998) 247–251. [12] T. Haaf, E.I. Golub, G. Reddy, C.M. Radding, D.C. Ward, Nuclear foci of mammalian Rad51 recombination protein in somatic cells after DNA damage and its localization in synaptonemal complexes, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 2298–2302. [13] S. Tashiro, J. Walter, A. Shinohara, N. Kamada, T. Cremer, Rad51 accumulation at sites of DNA damage and in postreplicative chromatin, J. Cell Biol. 150 (2000) 283–291. [14] E. Raderschall, E.I. Golub, T. Haaf, Nuclear foci of mammalian recombination proteins are located at single-stranded DNA regions formed after DNA damage, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 1921–1926. [15] T.L. Tan, J. Essers, E. Citterio, S.M. Swagemakers, J. de Wit, F.E. Benson, J.H. Hoeijmakers, R. Kanaar, Mouse Rad54 affects DNA conformation and DNA-damage-induced Rad51 foci formation, Curr. Biol. 9 (1999) 325–328. [16] Y. Liu, N. Maizels, Coordinated response of mammalian Rad51 and Rad52 to DNA damage, EMBO Rep. 1 (2000) 85–90. [17] E. Raderschall, A. Bazarov, J. Cao, R. Lurz, A. Smith, W. Mann, H.H. Ropers, J.M. Sedivy, E.I. Golub, E. Fritz, T. Haaf, Formation of higher-order nuclear Rad51 structures is functionally linked to p21 expression and protection from DNA damage-induced apoptosis, J. Cell Sci. 115 (2002) 153–164. [18] R.S. Tebbs, Y. Zhao, J.D. Tucker, J.B. Scheerer, M.J. Siciliano, M. Hwang, N. Liu, R.J. Legerski, L.H. Thompson, Correction of chromosomal instability and sensitivity to diverse mutagens by a cloned cDNA of the XRCC3 DNA repair gene, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 6354–6358. [19] J.S. Albala, M.P. Thelen, C. Prange, W. Fan, M. Christensen, L.H. Thompson, G.G. Lennon, Identification of a novel human RAD51 homolog, RAD51B, Genomics 46 (1997) 476–479. [20] R. Cartwright, C.E. Tambini, P.J. Simpson, J. Thacker, The XRCC2 DNA repair gene from human and mouse encodes a novel member of the recA/RAD51 family, Nucl. Acids Res. 26 (1998) 3084–3089. [21] M.K. Dosanjh, D.W. Collins, W. Fan, G.G. Lennon, J.S. Albala, Z. Shen, D. Schild, Isolation and characterization of RAD51C, a new human member of the RAD51 family of related genes, Nucl. Acids Res. 26 (1998) 1179–1184.

[22] N. Liu, J.E. Lamerdin, R.S. Tebbs, D. Schild, J.D. Tucker, M.R. Shen, K.W. Brookman, M.J. Siciliano, C.A. Walter, W. Fan, L.S. Narayana, Z.Q. Zhou, A.W. Adamson, K.J. Sorensen, D.J. Chen, N.J. Jones, L.H. Thompson, XRCC2 and XRCC3, new human Rad51-family members, promote chromosome stability and protect against DNA cross-links and other damages, Mol. Cell 1 (1998) 783–793. [23] D.L. Pittman, L.R. Weinberg, J.C. Schimenti, Identification, characterization, and genetic mapping of Rad51d, a new mouse and human RAD51/RecA-related gene, Genomics 49 (1998) 103–111. [24] J. Thacker, A surfeit of RAD51-like genes? Trends Genet. 15 (1999) 166–168. [25] C.S. Griffin, P.J. Simpson, C.R. Wilson, J. Thacker, Mammalian recombination-repair genes XRCC2 and XRCC3 promote correct chromosome segregation, Nat. Cell Biol. 2 (2000) 757–761. [26] M. Takata, M.S. Sasaki, E. Sonoda, T. Fukushima, C. Morrison, J.S. Albala, S.M. Swagemakers, R. Kanaar, L.H. Thompson, S. Takeda, The Rad51 paralog Rad51B promotes homologous recombinational repair, Mol. Cell. Biol. 20 (2000) 6476–6482. [27] M. Takata, M.S. Sasaki, S. Tachiiri, T. Fukushima, E. Sonoda, D. Schild, L.H. Thompson, S. Takeda, Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs, Mol. Cell. Biol. 21 (2001) 2858–2866. [28] J.P. Braybrooke, K.G. Spink, J. Thacker, I.D. Hickson, The RAD51 family member, RAD51L3, is a DNA-stimulated ATPase that forms a complex with XRCC2, J. Biol. Chem. 275 (2000) 29100–29106. [29] D. Schild, Y.C. Lio, D.W. Collins, T. Tsomondo, D.J. Chen, Evidence for simultaneous protein interactions between human Rad51 paralogs, J. Biol. Chem. 275 (2000) 16443–16449. [30] J.Y. Masson, M.C. Tarsounas, A.Z. Stasiak, A. Stasiak, R. Shah, M.J. McIlwraith, F.E. Benson, S.C. West, Identification and purification of two distinct complexes containing the five RAD51 paralogs, Genes Dev. 15 (2001) 3296–3307. [31] S. Sigurdsson, S. Van Komen, W. Bussen, D. Schild, J.S. Albala, P. Sung, Mediator function of the human Rad51B-Rad51C complex in Rad51/RPA-catalyzed DNA strand exchange, Genes Dev. 15 (2001) 3308–3318. [32] A.J. Pierce, R.D. Johnson, L.H. Thompson, M. Jasin, XRCC3 promotes homology-directed repair of DNA damage in mammalian cells, Genes Dev. 13 (1999) 2633–2638. [33] R.D. Johnson, N. Liu, M. Jasin Mammalian, XRCC2 promotes the repair of DNA double-strand breaks by homologous recombination, Nature 401 (1999) 397–399. [34] C.A. French, J.Y. Masson, C.S. Griffin, P.O. Regan, S.C. West, J. Thacker, Role of mammalian RAD51L2 (RAD51C) in recombination and genetic stability, J. Biol. Chem. 277 (2002) 19322– 19330. [35] B.C. Godthelp, W.W. Wiegant, A. van Duijn Goedhart, O.D. Scharer, P.P. van Buul, R. Kanaar, M.Z. Zdzienicka, Mammalian Rad51C contributes to DNA cross-link resistance, sister chromatid cohesion and genomic stability, Nucl. Acids Res. 30 (2002) 2172– 2182. [36] M.S. Meyn High, spontaneous intrachromosomal recombination rates in ataxia-telangiectasia, Science 260 (1993) 1327–1330. [37] R.H. Stauber, K. Horie, P. Carney, E.A. Hudson, N.I. Tarasova, G.A. Gaitanaris, G.N. Pavlakis, Development and applications of enhanced green fluorescent protein mutants, Biotechniques 24 (1998) 462–66, 468–471 [38] R.L. Capizzi, J.W. Jameson, A table for the estimation of the spontaneous mutation rate of cells in culture, Mutat. Res. 17 (1972) 147–148. [39] R.S. Cole, Repair of interstrand cross-links in DNA induced by psoralen plus light, Yale J. Biol. Med. 46 (1973) 492. [40] N. Magana Schwencke, J.A. Henriques, R. Chanet, E. Moustacchi, The fate of 8-methoxypsoralen photo-induced cross-links in nuclear and mitochondrial yeast DNA: comparison of wild-type and repair-deficient strains, Proc. Natl. Acad. Sci. U.S.A. 79 (1982) 1722–1726.

G.A. Drexler et al. / DNA Repair 3 (2004) 1335–1343 [41] I.U. De Silva, P.J. McHugh, P.H. Clingen, J.A. Hartley, Defining the roles of nucleotide excision repair and recombination in the repair of DNA interstrand cross-links in mammalian cells, Mol. Cell. Biol. 20 (2000) 7980–7990. [42] R.D. Johnson, M. Jasin, Double-strand-break-induced homologous recombination in mammalian cells, Biochem. Soc. Trans. 29 (2001) 196–201. [43] N. Liu, D. Schild, M.P. Thelen, L.H. Thompson, Involvement of Rad51C in two distinct protein complexes of Rad51 paralogs in human cells, Nucl. Acids Res. 30 (2002) 1009–1015. [44] K.A. Miller, D.M. Yoshikawa, I.R. McConnell, R. Clark, D. Schild, J.S. Albala, RAD51C interacts with RAD51B and is central to a larger protein complex in vivo exclusive of RAD51, J. Biol. Chem. 277 (2002) 8406–8411. [45] C. Wiese, D.W. Collins, J.S. Albala, L.H. Thompson, A. Kronenberg, D. Schild, Interactions involving the Rad51 paralogs Rad51C and XRCC3 in human cells, Nucl. Acids Res. 30 (2002) 1001–1008. [46] Y.C. Lio, A.V. Mazin, S.C. Kowalczykowski, D.J. Chen, Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro, J. Biol. Chem. 278 (2003) 2469– 2478. [47] C.M. Luo, W. Tang, K.L. Mekeel, J.S. DeFrank, P.R. Anne, S.N. Powell, High frequency and error-prone DNA recombination in ataxia telangiectasia cell lines, J. Biol. Chem. 271 (1996) 4497–4503.

1343

[48] E. Fritz, A.A. Friedl, R.M. Zwacka, F. Eckardt Schupp, M.S. Meyn, The yeast TEL1 gene partially substitutes for human ATM in suppressing hyperrecombination, radiation-induced apoptosis and telomere shortening in A-T cells, Mol. Biol. Cell 11 (2000) 2605–2616. [49] S. Sengupta, S.P. Linke, R. Pedeux, Q. Yang, J. Farnsworth, S.H. Garfield, K. Valerie, J.W. Shay, N.A. Ellis, B. Wasylyk, C.C. Harris, BLM helicase-dependent transport of p53 to sites of stalled DNA replication forks modulates homologous recombination, EMBO J. 22 (2003) 1210–1222. [50] P.R. Prince, M.J. Emond, R.J. Monnat, Loss of Werner syndrome protein function promotes aberrant mitotic recombination, Genes Dev. 15 (2001) 933–938. [51] M.E. Moynahan, J.W. Chiu, B.H. Koller, M. Jasin, Brca1 controls homology-directed DNA repair, Mol. Cell 4 (1999) 511–518. [52] M.E. Moynahan, A.J. Pierce, M. Jasin, BRCA2 is required for homology-directed repair of chromosomal breaks, Mol. Cell 7 (2001) 263–272. [53] F. Xia, D.G. Taghian, J.S. DeFrank, Z.C. Zeng, H. Willers, G. Iliakis, S.N. Powell, Deficiency of human BRCA2 leads to impaired homologous recombination but maintains normal nonhomologous end joining, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 8644–8649. [54] E. Raderschall, K. Stout, S. Freier, V. Suckow, S. Schweiger, T. Haaf, Elevated levels of Rad51 recombination protein in tumor cells, Cancer Res. 62 (2002) 219–225.