Cooperative Roles of Vertebrate Fbh1 and Blm DNA Helicases in Avoidance of Crossovers during Recombination Initiated by Replication Fork Collapse

Fbh1 (F-box DNA helicase 1) orthologues are conserved from Schizosaccharomycespombe to chickens and humans. Here, we report the disruptionof the FBH1 gene in DT40 cells. Although the yeast fbh1 mutantshows an increase in sensitivity to DNA damaging agents, FBH1^–^/^–DT40 clones show no prominent sensitivity, suggesting that theloss of FBH1 might be compensated by other genes. However, FBH1^–^/^–cells exhibit increases in both sister chromatid exchange andthe formation of radial structures between homologous chromosomeswithout showing a defect in homologous recombination. This phenotypeis reminiscent of BLM^–^/^– cells and suggests thatFbh1 may be involved in preventing extensive strand exchangeduring homologous recombination. In addition, disruption ofRAD54, a major homologous recombination factor in FBH1^–^/^–cells, results in a marked increase in chromosome-type breaks(breaks on both sister chromatids at the same place) followingreplication fork arrest. Further, FBH1BLM cells showed additiveincreases in both sister chromatid exchange and the formationof radial chromosomes. These data suggest that Fbh1 acts inparallel with Bloom helicase to control recombination-mediateddouble-strand-break repair at replication blocks and to reducethe frequency of crossover.

INTRODUCTION

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Introduction

DNA damage is continuously generated by cellular metabolic productsas well as by environmental factors (23) and poses a seriousthreat to the cells. In cycling cells, it may inhibit the progressionof replication forks, resulting in gaps and occasionally double-strandbreaks (DSBs) in the daughter strands (4, 34). To release replicationblocks and thereby prevent DSBs, cells have evolved two majorrepair pathways, translesion DNA synthesis and homologous recombination(HR) (15). Translesion synthesis is carried out by a numberof specialized translesion synthesis DNA polymerases, whichare able to synthesize directly across damaged DNA bases. Asan alternative to translesion synthesis, bypass can be effectedby a form of HR involving a transient template switch from damagedstrand to the newly synthesized daughter strand on the sisterchromatid (16). HR is also required for repair of DSBs arisingat collapsed replication forks, caused by DNA-damaging agentssuch as the DNA topoisomerase I inhibitor camptothecin (1, 14).This chemotherapeutic agent covalently attaches to topoisomeraseI, allowing it to cleave but not religate DNA. Upon encounterwith a replication fork, the topoisomerase I-cleaved DNA complexinduces a single-end break (4, 8, 37). Restart of replicationfrom these single-end breaks requires HR with the intact sisterchromatid.

The initial step of recombination-mediated DSB repair involvesprocessing of the DSBs to produce a 3' single-strand overhang,followed by polymerization of Rad51 on the single-stranded DNA.In concert with Rad54 (2, 31, 38), the resulting nucleoproteinfilament facilitates the homology search and pairing with theintact duplex DNA donor to form a D-loop structure. FollowingDNA synthesis from the invading 3' end, the strand is dissociatedfrom the D loop and rehybridizes with the 3' single-strand tailof the other side of the DSB. This type of HR-dependent DSBrepair is called synthesis-dependent strand annealing, whichis thought to account for a majority of mitotic HR (18, 30).In contrast to synthesis-dependent strand annealing, which doesnot result in the formation of crossovers, strand exchange followingD-loop formation results in the formation of a Holliday junction.Holliday junctions can be resolved by incisions at the junction,which can result in crossover formation (24). It has also beenproposed that double Holliday junctions are dissolved by theconcerted action of the RecQ helicase Blm and topoisomeraseIII ${alpha}$ (42).

The BLM helicase is mutated in Bloom's syndrome. This rare autosomalrecessive genetic disease features an elevated rate of sisterchromatid exchange (SCE), frequent recombination between allelicgenes, increased genome instability, and a high incidence oftumorigenesis (9). The Bloom helicase is one of five human RecQhelicases, which share 3'-to-5' DNA helicase activity as wellas structural homology. Yeast species, on the other hand, haveonly a single orthologous gene, Sgs1 in Saccharomyces cerevisiaeand Rqh1 in Schizosaccharomyces pombe (12). Although these RecQfamily helicases have been implicated in the initiation of replicationrestart without crossover and resolution of recombination intermediates(32, 40, 42), their function remains poorly understood evenin yeast due to the complexity of repair reactions followingreplication block and the lack of an appropriate phenotypicassay to monitor the late steps of HR in vivo. Likewise, thedetailed molecular function of the mammalian RecQ helicase remainselusive.

The FBH1 (F-box DNA helicase 1) gene was recently identifiedin Schizosaccharomyces pombe, and its orthologous genes areconserved in chickens, mice, and humans but not in budding yeast(22). Fbh1 belongs to the superfamily 1 helicase and has unwindingactivity with a 3'-to-5' direction. Fbh1 is structurally similarto the Srs2 DNA helicase family, which includes the PcrA, Rep,and UvrD helicases (43). Fbh1 also contains an F box towardits N terminus, suggesting that Fbh1 acts as a ubiquitin ligase(E3) in the SCF complex, although the substrate of the ubiquitinligase has not been determined (21). The S. pombe fbh1 mutantis moderately sensitive to UV; methylmethane sulfonate (MMS),an alkylating agent; and ${gamma}$ -rays. Genetic studies show that Fbh1is essential for viability in the absence of Rqh1 and that thislethality is suppressed by additional inactivation of Rad51paralogs, which promote an early step of HR. Thus, Fbh1 mayaffect recombination-dependent repair downstream of Rhp51 (S.pombe Rad51 ortholog), probably by promoting the processingof recombination intermediates. This notion is supported bythe finding that spontaneous Rad51 focus formation was enhancedin an fbh1-deficient S. pombe mutant (27, 29). Therefore, Fbh1may share a redundant function with Srs2 in S. pombe.

In this study, we established FBH1-deficient DT40 cells to investigateFbh1 function in vertebrate cells. The resulting FBH1^–^/^–cells exhibited essentially a normal phenotype. The functionaloverlap between yeast Fbh1 and Rqh1 led us to investigate whetherother genes compensate for the loss of Fbh1 in DT40 cells. Tothis end, we disrupted the FBH1 gene in DT40 lines mutant forthe FANCC^–, RAD18^–^/^–, RAD54^–^/^–,and BLM^–^/^– genes (5, 13, 17, 44). We show that althoughinactivation of FBH1 did not reduce the efficiency of HR, Fbh1acts in parallel with Blm and restricts the extent of strandexchange during recombination initiated by stalled replication,thereby reducing the frequency of crossover formation.

MATERIALS AND METHODS

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Materials and Methods

Cloning of the chicken FBH1 gene. A chicken FBH1 cDNA was partially amplified from chicken testiscDNA by reverse transcriptase PCR (RT-PCR) with the primer pair5'-AAATCATGGCTTTTGCNGGNACTGG-3' and 5'-CACCTCGGAAGGAATAGATCTGCTG-3',the design of which was based on conserved sequences betweenthe human and xenopus FBH1 homologs. Rapid amplification ofcDNA ends was carried out to isolate the 3' and 5' termini ofthe chicken FBH1 transcript. Full-length cDNA of chicken FBH1(GdFBH1) was amplified from chicken testis cDNA by RT-PCR witha primer pair, 5'-AGAAAATGCACCTTACAGCTG-3' and 5'-GCAATGGCTCTGGGAGTTGG-3'.An amplified 3-kb fragment was subcloned into a pCR2.1-TOPOvector (Invitrogen) and sequenced.

Construction of targeting vectors. The positions of exons and introns were determined by base sequencing.Genomic DNA sequences were amplified with two sets of primers,5'-CTGCCACAGATATGCTAGAG-3' and 5'-TTTCCCATGTACTCATCCTC-3' plus5'-GACTTTGTGGATGTACCGTG-3' and 5'-ATGGAGCCAAAACGCTTCTG-3', forthe left and right arms, respectively, of the disruption construct.Amplified PCR products (1.5 kb for the left arm and 3.5 kb forthe right arm) were cloned into pCR2.1-TOPO vector (Invitrogen).The 3.5-kb KpnI (blunt ended)-NotI fragment was cloned intothe EcoRV-NotI site of pCR2.1 containing the 1.5-kb left armsequence. The BamHI site was used to insert marker gene cassettes,hisD and bsr selection marker genes flanked by loxP sequences,to generate the FBH1-hisD and FBH1-bsr gene disruption constructs,respectively (3). The puro marker gene was inserted into theBamHI site to generate the FBH1-puro gene disruption construct.The 0.5-kb fragment generated by PCR amplification of genomicDNA using the primers 5'-TTCAGGAAGAGCCCATCGTG-3' and 5'-AATGTCCCCACTGAAACAGG-3'was used as a probe for Southern blot analysis to screen gene-targetingevents. Plasmids were linearized prior to transfection intoDT40 cells; FBH1-hisD and FBH1-bsr were digested with XhoI,and FBH1-puro was digested with KpnI.

Generation of gene-disrupted cells. Wild-type DT40 cells that stably express the CreER chimericrecombination enzyme were transfected sequentially with FBH1-bsrand FBH1-hisD targeting constructs to obtain FBH1^+/^– andFBH1^–^/^– cells (Fig. 1A) (10, 25). Stable expressionof CreER did not affect the viability of the cells or theirsensitivity to DNA damaging agents (data not shown). Targetingevents were verified by the appearance of a 6-kb band and thedisappearance of a 9-kb band in Southern blot analysis of EcoRI-digestedgenomic DNA (Fig. 1B). Gene disruption of each transfected clonewas confirmed by RT-PCR analysis of FBH1 gene transcription.The primers used for RT-PCR were the forward primer 5'-TTGAGCGTGGTCAGATAGTG-3'and the reverse primer 5'-CACCTCGGAAGGAATAGATCTGCTG-3' (Fig.1D). ß-Actin transcripts were analyzed as positivecontrols for RT-PCR analysis. Identified FBH1^–^/^–clones were treated with 100 nM tamoxifen to exclude bsr andhisD marker cassettes from chromosomal DNA (44). The resultingFBH1^–^/^– cells were sequentially transfected withBLM-hisD and BLM-bsr targeting constructs to obtain FBH1^–^/^–BLM^+/^– and FBH1^–^/^– BLM^–^/^– clones,respectively (41). Likewise, RAD54-hisD and RAD54-puro targetingconstructs were used for generating FBH1^–^/^– RAD54^–^/^–cells (5). The FANCC gene, which is encoded in a single sexchromosome, was disrupted in FBH1^–^/^– cells by usingthe FANCC-puro plasmid (13). RAD18^–^/^– cells weresequentially transfected with FBH1-bsr and FBH1-puro targetingconstructs to obtain FBH1^–^/^– RAD18^+/^– andFBH1^–^/^– RAD18^–^/^– clones, respectively(44). To express chicken Fbh1, its cDNA was inserted into thep176 expression vector containing the Neo selection marker gene(10). The expression plasmid was linearized with PvuI priorto transfection into cells. The conditions for cell culture,selection, and DNA transfections have been described previously(35).

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FIG. 1. Gene targeting of the FBH1 locus. (A) Schematic representation of a part of the FBH1 locus and the targeting constructs. The FBH1 replacement constructs, which contain a bsr or hisD selection marker gene, were used to disrupt the FBH1 locus. The filled box represents the FBH1 exons. RI, EcoRI. (B) Southern blot analysis of EcoRI-digested DNA using the probe indicated in panel A. (C) RT-PCR analysis of total RNA using the primers shown in panel A. The coding region of the chicken ß-actin gene was amplified as a control. (D) Normal growth kinetics of FBH1^–^/^– cells. (E) Cell cycle distribution of the indicated cell lines. Cells were stained with FITC-anti-BrdU antibody (y axis, log scale) to detect BrdU uptake and with propidium iodide to measure the total DNA (x axis, linear scale). The upper gate identifies cells incorporating BrdU ( $~$ S phase), the lower left gate identifies G₁ cells, and the lower right gate displays G₂/M cells. The sub-G₁ fraction reflects dead cells. The numbers given in the boxes indicate the percentages of gated events.

Proliferation analysis and colony formation assay. A colony formation assay was performed as described previously(28). To measure the sensitivity to camptothecin, cells wereplated in methylcellulose medium containing camptothecin. Theexperimental methods for cell counting and cell cycle analysiswere described previously (39).

Measurement of SCE levels. Measurement of SCE levels was carried out as described previously(28, 44). To measure camptothecin-induced SCE, cells were labeledwith bromodeoxyuridine (BrdU) for two cell cycle periods ( $~$ 20h) and treated with 5 nM camptothecin for the last 9 h. To measureSCE induced by UV, cells were treated with 0.25 J/m² of UV andincubated with 10 uM BrdU for 20 h. The cells were incubatedwith Colcemid to enrich metaphase cells for the last 3 h beforeharvest.

Chromosomal aberration analysis. Preparation of chromosome spreads and karyotype analysis wereperformed as described previously (35, 44). To measure camptothecin-inducedchromosomal aberrations, cells were incubated with 10 nM camptothecinfor 9 h before harvest and fixation. To analyze UV-induced chromosomalaberrations, cells were suspended in 1 ml of phosphate-bufferedsaline, spread onto six-well plates, and exposed to 1 J/m² UVC,followed by the addition of 5 ml of the complete medium. Cellswere harvested every 3 h for 15 h. To enrich mitotic cells,cells were exposed to Colcemid for the last 3 h.

Measurement of targeted integration frequencies. HR was evaluated by the following three methods. First, thefrequency of I-SceI restriction enzyme-induced HR was measuredas described previously (11, 19). The assay plasmid was insertedinto the OVALBUMIN locus. Second, to analyze targeted integrationevents at the OVALBUMIN, RAD54, and CENP-H loci, each disruptionconstruct was transfected into cells. Gene targeting eventswere identified by either Southern blot analysis or FACS analysisof EGFP-CENP-H fusion protein expression (20). Lastly, HR-mediatedimmunoglobulin V (IgV) diversification (Ig gene conversion)was monitored, as described previously (6). To this end, FBH1^–^/^–clones were generated from wild-type DT40 cells that carry aspecific frameshift mutation. The rate of Ig gene conversionwas assessed by measuring the gain of surface IgM expressionduring the 3-week period with clonal expansion.

Nucleotide sequence accession number. The chicken FBH1 cDNA sequences have been submitted to the GenBankdatabase under accession number EF066526.

RESULTS

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Results

No apparent defect in HR in the absence of Fbh1. The chicken FBH1 encodes a polypeptide comprised of 1,012 aminoacids that has 55%, 50%, and 26% identity at the amino acidlevel to the human, mouse, and S. pombe orthologs, respectively.Gene-targeting constructs were generated to delete amino acids118 to 875, including both the F-box domain and five helicasemotifs (Ia, Ib, II, III, IV) of the FBH1 gene (Fig. 1A; alsosee Fig. S1 in the supplemental material). Two independent FBH1^–^/^–clones were identified by Southern blot analysis (Fig. 1B).Gene disruption of these clones was verified by RT-PCR (Fig.1C). FBH1^–^/^– cells were able to proliferate withnormal kinetics (Fig. 1D). Flow cytometric analysis of BrdUpulse-labeled cells showed a normal cell cycle distribution(Fig. 1E).

We evaluated the capability of HR in FBH1^–^/^– cellsby using the following phenotypic assays: sensitivity to DNAdamaging agents (Fig. 2), gene-targeting frequency (Table 1),HR-mediated repair of DSBs induced by the I-SceI restrictionenzyme, HR-dependent diversification of the Ig gene (Ig geneconversion), and the kinetics of Rad51 subnuclear focus formation.FBH1^–^/^– cells were tolerant to ${gamma}$ -rays, MMS, UV, andcisplatin, as were wild-type cells (Fig. 2A to D), while exhibitingmodest sensitivity to camptothecin (Fig. 2E). The efficiencyof both gene targeting (Table 1) and HR-dependent DSB repairfollowing I-SceI expression (Fig. 3A) was indistinguishablebetween wild-type and FBH1^–^/^– cells. Likewise, FBH1^–^/^–cells appear to undergo Ig gene conversion with normal kinetics(data not shown). The kinetics of subnuclear Rad51 focus formationfollowing ${gamma}$ -irradiation were the same between wild-type and FBH1^–^/^–cells during the cell cycle and at 3 h, 6 h (Fig. 3B), and 24h (data not shown) after ${gamma}$ -irradiation. Collectively, FBH1^–^/^–cells can perform HR reactions with normal kinetics, which agreeswith the previous finding of no effect on spontaneous and DNAdamage-induced recombination in Fbh1-deficient yeast (29).

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FIG. 2. Sensitivities of the FBH1 mutant to genotoxic stresses. The indicated genotypes of cells were exposed to ${gamma}$ -rays (A) and UV (B), after a 1-h treatment with MMS (C) and cisplatin (D) and after continuous exposure to camptothecin (CPT) (E). The doses of genotoxic agents are displayed on the x axes on a linear scale, while the fractions of surviving colonies are displayed on the y axes on a logarithmic scale. Error bars show the standard error of the mean for at least three independent experiments.

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TABLE 1. Targeted integration frequencies^a

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FIG. 3. No significant defect in HR in FBH1^–^/^– cells. (A) I-SceI-induced gene conversion assay with the indicated genotypes. The frequency of HR-dependent double-strand-break repair is shown as the number of G418-resistant colonies per 5 x 10⁶ cells (white bars). The experiments were done at least three times. (B) Rad51 focus formation with the indicated genotypes before and at 3 and 6 h after 4 Gy ${gamma}$ -irradiation. (C) Spontaneous and 5 nM camptothecin (CPT)-induced SCE events in the macrochromosomes of 50 metaphase cells. Means ± standard errors are shown in the upper right corner. Underlined numbers indicate subtraction of spontaneous SCE from SCE following CPT treatment. The level of spontaneous SCE in FBH1^–^/^– cells significantly differs from that in wild-type cells (P < 0.0001). The level of CPT-induced SCE was indistinguishable between wild-type and FBH1^–^/^– cells (P < 0.0001). Statistical significance was calculated by the Mann-Whitney nonparametric U test (36). (D) UV-induced SCE (0.25 J/m²) is shown as in panel C. The level of UV-induced SCE was indistinguishable between wild-type and FBH1^–^/^– cells (P < 0.0004).

Deletion of the FBH1 gene has no significant effect on FANCC- or RAD18-deficient cells. Despite apparently normal HR, FBH1^–^/^– cells exhibitedsignificant increases in the level of spontaneously arisingSCE (Fig. 3C and D). This increased SCE was reversed by reconstitutionof FBH1^–^/^– cells with chicken FBH1 cDNA (data notshown). Taking the normal HR efficiency of FBH1^–^/^–cells into account, the increased spontaneous SCE suggests eitheran increase in the amount of endogenous DNA damage that stimulatesSCE or an elevated ratio of crossover-type HR relative to non-crossover-typeHR. To gain an insight into the increased spontaneous SCE, wemeasured SCE induced by camptothecin and UV (Fig. 3C and D),which result in DSBs and single-strand gaps, respectively, whenreplication forks encounter the damaged sites. Subtraction ofspontaneous SCE from camptothecin-induced SCE indicates thatcamptothecin-induced SCE was at the same level in wild-typeand FBH1^–^/^– cells (Fig. 3C). In contrast, UV induceda moderately higher level of SCE in FBH1^–^/^– cellsthan in wild-type cells (Fig. 3D). Thus, we suggest that single-strandgaps spontaneously formed at stalled replication forks stimulatecrossover-type HR more frequently in the absence of Fbh1. Further,camptothecin-induced DSBs may not be repaired adequately byHR and may lead to cell death in FBH1^–^/^– cells (Fig.2E).

The elevation of spontaneous SCE led us to explore the functionalinteractions between Fbh1 and either FancC or Rad18, becausecells deficient in these proteins exhibit increased levels ofspontaneous SCE (13, 44). To this end, we made FBH1^–^/^–FANCC^– and FBH1^–^/^– RAD18^–^/^– DT40cells. Inactivation of FBH1 had no significant impact on FANCC^–and RAD18^–^/^– cells in terms of cellular sensitivityto cisplatin and MMS (see Fig. S2A and C and Fig. S3A in thesupplemental material; also data not shown). On the other hand,FBH1^–^/^– RAD18^–^/^– cells exhibited slightlyhigher UV sensitivity than did RAD18^–^/^– cells (seeFig. S2D in the supplemental material). In addition, camptothecinsensitivity and spontaneous SCE were increased in FBH1^–^/^–RAD18^–^/^– and FBH1^–^/^– FANCC^– cellscompared with each single mutant (see Fig. S2B and S3B in thesupplemental material; also data not shown). These observationsindicate no significant functional overlap between Fbh1 andeither Rad18 or FancC in suppression of SCE.

Synergistic effect of defective FBH1 and RAD54 on genome instability and hypersensitivity to camptothecin. Given that the SCE phenotype of FBH1^–^/^– cells suggestedthat recombination events more frequently resulted in crossover,we sought to test the relationship between Fbh1 and the corerecombination factor Rad54 (5). FBH1^–^/^– RAD54^–^/^–cells grew more slowly, displaying a significant fraction ofdying cells compared to each single mutant (Fig. 1E and 4A).Likewise, in FBH1^–^/^– RAD54^–^/^– cells,higher fractions in the sub-G₁ and G₂ phases were elevated incomparison to the FBH1^–^/^– and RAD54^–^/^–single mutants (Fig. 1E). As expected, the level of spontaneousSCE was reduced by deletion of the RAD54 gene in FBH1^–^/^–cells (data not shown). Collectively, deletion of FBH1 in aRAD54^–^/^– background compromises genome stability,leading to stimulation of a G₂ damage checkpoint and apoptosis.

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FIG. 4. FBH1^–^/^– RAD54^–^/^– cells display genome instability and hypersensitivity against camptothecin (CPT). (A) Growth curves of cells of the indicated genotypes. Results were obtained from at least three independent experiments. (B and C) Colony survival assay after treatment with CPT and cisplatin performed as described for Fig. 2. Error bars show the standard error of the mean for at least three independent experiments. (D) Chromosome aberrations before and following 10 nM CPT treatment for 9 h with the indicated genotypes.

FBH1^–^/^– RAD54^–^/^– cells were more sensitiveto camptothecin than was either single mutant (Fig. 4B). Onthe other hand, cellular sensitivity to cisplatin (Fig. 4C), ${gamma}$ -rays, and MMS (data not shown) was indistinguishable betweenRAD54^–^/^– and FBH1^–^/^– RAD54^–^/^–cells. To assess the cause of cellular hypersensitivity to camptothecin,chromosomal aberrations were monitored following exposure ofcells to camptothecin (Fig. 4D). In agreement with colony survivaldata, the FBH1^–^/^– RAD54^–^/^– cells exhibiteda marked increase in camptothecin-induced chromosomal aberrationscompared to RAD54^–^/^– cells (Fig. 4D). The numberof chromosomal aberrations, as well as camptothecin sensitivity,was reversed by ectopic expression of chicken FBH1 cDNA (GdFBH1)in FBH1^–^/^– RAD54^–^/^– cells (Fig. 4B and D).It should be noted that camptothecin-induced chromosome-typebreaks (DSBs at the same position on two sister chromatids)were significantly increased in FBH1^–^/^– RAD54^–^/^–cells in comparison with RAD54^–^/^– cells. Since camptothecininduces DSBs in one of the two sister chromatids and subsequentlystimulates HR with the other intact sister, DSBs at the sameposition in two sister chromatids, i.e., chromosome-type breaks,indicate that sister recombination is not efficiently completedin FBH1^–^/^– RAD54^–^/^– cells, leading toDSBs in both sister chromatids during the S and G₂ phases (35).

This raises two hypotheses concerning the function of Fbh1 inDSB repair. First, both Fbh1 and Rad54 may be involved in thesame stage of HR, with Fbh1 acting as a backup for Rad54-dependentHR. Alternatively, Fbh1 may prevent the formation of abnormalrecombination intermediates, which lead to chromosome-type breaksin the absence of Rad54. To test the former hypothesis, we assayedHR in RAD54^–^/^– and FBH1^–^/^– RAD54^–^/^–cells by monitoring the repair of I-SceI induced DSBs. Deletionof FBH1 did not further diminish the capability of HR in RAD54^–^/^–cells (Fig. 3A). In addition, Rad51 focus formation following ${gamma}$ -irradiation and camptothecin was comparable in RAD54^–^/^–and FBH1^–^/^– RAD54^–^/^– clones (data notshown). These data are consistent with disruption of FBH1 resultingin the formation of recombination intermediates that are preferentiallyresolved with crossover and which lead to chromosome-type breaksin the absence of Rad54.

Complementary role of Fbh1 and Blm in releasing the DNA replication block. The synthetic lethality of the fbh1 and rqh1 mutations in yeast,as well as an increase in the level of spontaneous SCE in Fbh1-deficientDT40 clones, prompted us to disrupt the BLM gene in FBH1^–^/^–cells (9, 17, 41). Unlike in S. pombe, FBH1^–^/^– BLM^–^/^–DT40 cells were able to proliferate with the same kinetics asBLM^–^/^– cells (Fig. 5A), but FBH1^–^/^–BLM^–^/^– cells were considerably more sensitive tocamptothecin than was either single mutant (Fig. 5B). Conversely,the sensitivity of FBH1^–^/^– BLM^–^/^– cellsto UV, MMS, and cisplatin was the same as that of BLM^–^/^–cells (Fig. 5C and D and data not shown). Thus, Fbh1 may substitutefor the loss of Blm, particularly when DSBs occur as a consequenceof a replication block.

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FIG. 5. Combined mutations of the FBH1 and BLM genes synergistically elevate sensitivity to camptothecin (CPT). (A) Growth curves of cells of the indicated genotypes. (B to D) Colony survival assay after treatment with CPT, UV, and cisplatin performed as described for Fig. 2. Error bars show the standard error of the mean for at least three independent experiments.

To further explore the functional overlap between Fbh1 and Blm,we examined SCE and mitotic exchanges between homologous chromosomes,because a significant increase in these two types of crossoversis the characteristic feature of BLM-deficient cells (17, 41).FBH1^–^/^– and BLM^–^/^– DT40 cells showed1.8- and 9.4-fold-higher numbers of spontaneous SCE events thandid wild-type cells, respectively (Fig. 6A). The FBH1^–^/^–BLM^–^/^– cells displayed further increases in thelevel of spontaneous SCE. We next examined the level of UV-inducedSCE (Fig. 6B). The induction of SCE tended to be higher in FBH1^–^/^–and FBH1^–^/^– BLM^–^/^– cells than in wild-typeand BLM^–^/^– cells, respectively (Fig. 6B); the numberof SCEs in a UV-irradiated BLM^–^/^– cell is likelyto be saturated, making definitive quantitative measurementsunreliable.

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FIG. 6. Increased genome instability of FBH1^–^/^– BLM^–^/^– cells observed in both UV-induced SCE and exchange between homologous chromosomes. Cells were labeled with BrdU during two cell cycle periods with (B) or without (A) prior UV treatment (0.25 J/m²). Spontaneous and UV-induced SCEs in the macrochromosomes of 50 metaphase cells were counted. Underlined numbers indicate subtraction of spontaneous SCE from SCE following UV. Histograms show the distribution of cells with the indicated numbers of SCEs per cell. The mean number of SCEs per cell ± standard error is shown in each histogram. The level of spontaneous SCE in BLM^–^/^– cells significantly differs from that in FBH1^–^/^– BLM^–^/^– cells (P < 0.02). The level of UV-induced SCE was distinguishable between BLM^–^/^– and FBH1^–^/^– BLM^–^/^– cells (P < 0.03), though it may have reached a plateau level. Statistical significance was calculated by the Mann-Whitney nonparametric U test. (C) Representative homologous chromosome exchange in FBH1^–^/^– BLM^–^/^– cells. Arrowheads indicate chromosome 1. (D) Example of nonhomologous exchange events between chromosomes 1 and 2 and between chromosomes 2 and 4 shown by arrowheads. (E) Frequency of chromosomal aberrations in mitotic cells harvested at indicated times after exposure of asynchronous cell populations to 1 J/m² UV. Chromosome aberrations were scored for 100 metaphase cells. All samples were treated with Colcemid for the last 3 h prior to harvest of cells.

We monitored mitotic chromosome aberrations with time followingUV irradiation (Fig. 6C to E). Mitotic cells were collectedevery 3 h from 0 to 15 h after 1 J/m² UVC irradiation of asynchronouscells. Cells entering metaphase at 0 to 6 h and 9 to 12 h afterUV irradiation reflect cells that received UV irradiation inthe late S to G₂ phase and the G₁ to early S phase, respectively(17). UV irradiation increased exchange between homologous chromosomes,i.e., symmetrical chromosomal quadriradials (Fig. 6C), but didnot increase exchange between different chromosomes (Fig. 6D)9 to 12 h after UV irradiation in asynchronous wild-type cells(i.e., cells that were exposed to UV in G₁ to early S phase).These chromosomal quadriradials may result from persisting Hollidayjunctions formed at sites of replication stalled by UV-inducedDNA damage. As expected, BLM^–^/^– cells exhibiteda marked increase in the number of homologous chromosome exchangesin comparison to wild-type cells (Fig. 6E). This type of chromosomalaberration depends on HR, because deletion of HR factors suchas RAD54 and XRCC3 in BLM^–^/^– cells reduced the formationof symmetrical chromosomal quadriradials by $~$ 10-fold (M. Seki,personal communication). Like BLM^–^/^– cells, FBH1^–^/^–cells displayed an increase in homologous chromosome exchange.Furthermore, concurrent deletion of FBH1 and BLM had an additiveeffect on UV-induced quadriradial formation compared to FBH1^–^/^–and BLM^–^/^– single mutant cells. The total numbersof chromosomal quadriradials were 15 (wild type), 23 (FBH1^–^/^–),56 (BLM^–^/^–), and 72 (FBH1^–^/^– BLM^–^/^–)per 600 metaphase cells in each genotype. These results suggesta possible involvement of the Fbh1 helicase, in parallel withthe Blm helicase, in resolving otherwise toxic recombinationintermediates produced at the sites of stalled replication forks.

DISCUSSION

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Discussion

Role for vertebrate Fbh1 during DNA replication. As shown in Fig. 7, there are some processes to restrict theextent of strand exchange during recombination produced at thesites of stalled replication forks, resulting in a reduced frequencyof crossover formation. Although several DNA helicases havebeen involved in these processes, the precise mechanism of thismodel remains to be determined. Several lines of evidence pointto Fbh1 being involved in preventing the formation of recombinationintermediates following replication blockage. The formationof such recombination intermediates (Fig. 7; transition fromC to E) could explain the phenotypes associated with the inactivationof Fbh1. None of the experimental data presented here indicatethat non-crossover-type HR, which accounts for the majorityof HR during somatic cell division in vertebrates (18), is increasedin the absence of FBH1. Neither gene targeting, HR-dependentrepair of I-SceI-induced DSBs, nor Ig gene conversion was decreasedin frequency in FBH1^–^/^– cells. Deletion of FBH1had no effect on sensitivity to UV, MMS, and cisplatin, whichblock replication and induce recombination, even in the absenceof BLM. Thus, it does not seem likely that Fbh1 processes putativerecombination intermediates (such as the chicken foot or doubleHolliday junction) directly. Despite a lack of evidence fora general increase in HR events in FBH1^–^/^– cells,FBH1 deficiency leads to a significant increase in the numberof crossovers between homologous chromosomes (radial structures)after UV irradiation. This, coupled with an increased levelof camptothecin-induced chromosome-type breaks in FBH1^–^/^–RAD54^–^/^– cells, indicates that sister-mediated HR-dependentrepair is blocked after physical interactions of two sisterchromatids in the absence of Fbh1. Such a defect in the suppressionof processing recombination intermediates in FBH1^–^/^–cells after DNA damage accords with the phenotype of S. pombeFbh1 cells (27). In conclusion, Fbh1 is likely to prevent extensiveexchange between two homologous sequences, thereby facilitatingHR-dependent repair of single-ended breaks formed during replication.

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FIG. 7. Model for the function of Fbh1 and Blm at stalled replication forks. A replication fork encounters damages on the template (oval) and is blocked. This block can be bypassed by translesion synthesis (transition from A to G), or the fork can collapse (transition from A to B). HR to restart the collapsed fork is usually carried out by synthesis-dependent strand annealing (SDSA), which is not associated with crossover (transition from C to D). More extensive strand exchange between sister chromatids (E) leads to the formation of a double Holliday junction, which may be resolved with crossover (F). Blm may play a number of roles that reduce crossovers. It may suppress D-loop formation (transition from B to C) (40), reverse Holliday junction formation (transition from C to E) (26) and, with topoisomerase III ${alpha}$ , promote the dissolution of double Holliday junctions, thereby avoiding resolution by incision (42). The present data support a role for Fbh1 acting alongside Blm in the second of these possibilities, the avoidance of extensive strand exchange and Holliday junction formation (transition from C to E).

Fbh1 and Blm act in parallel in the repair of DSBs that occur as a consequence of replication block. The present study reveals phenotypic similarities as well asdifferences in the mutant phenotype of the FBH1 orthologs ofDT40 and S. pombe. In both species, deletion of the FBH1 geneaugments the phenotype of BLM^–^/^–/rqh1 mutants. Onthe other hand, FBH1^–^/^– cells did not show a prominentphenotype, while S. pombe fbh1 mutants exhibit sensitivity tovariety of DNA damaging agents and enhanced Rad51 focus formation.Thus, the prominent role of Fbh1 in S. pombe is not seen inDT40, suggesting that either vertebrate cells have a reducedrequirement for this helicase or the functions of Fbh1 can becovered by other helicases as well as Blm. BLM-deficient cellsare characterized by hyper-recombination between sister chromatidsand also between homologous chromosomes (7). These phenotypeswere also observed, although to a lesser degree, in FBH1^–^/^–cells (Fig. 3C and D and 6E). It should be noted that the FBH1mutation increased the sensitivity of BLM^–^/^– cellsto camptothecin but not to any other damaging agents (Fig. 5B to D). Spontaneous single-strand nicks are likely to collapsereplication forks to produce single-ended breaks similar tothose induced by camptothecin (Fig. 7B) (33). These single-endedbreaks trigger recombination-mediated replication restart, hencea requirement for Rad54, explaining the additive compromisein genomic stability and sensitivity to camptothecin in theFBH1^–^/^– RAD54^–^/^– double mutant. Thus,the absence of Fbh1 or Blm may cause an accumulation of recombinationintermediates that normally require these DNA helicases fortheir resolution. The synergistic effect of disruption of RAD54in both FBH1^–^/^– and BLM^–^/^– cells providesfurther support for a functional similarity between Fbh1 andBlm (41).

Taking our results together, we conclude that the Fbh1-dependentpathway is likely to act in parallel to Blm in cellular toleranceof single-ended breaks resulting from replication blocks. Inhumans, Fbh1 may play a role in suppression of the loss of heterozygosity,like Blm, thereby preventing tumor formation. No human FBH1mutants have been reported, possibly because FBH1 disruptionalone does not result in a clinically detectable syndrome or,although less likely given the lack of a phenotype in DT40 cells,is lethal. However, it will be interesting to determine whethera defect in this helicase function is associated with cancerpredisposition or even a genetic disease akin to Bloom's andWerner's syndromes.

ACKNOWLEDGMENTS

We thank the members of the Takeda laboratories for help andsupport. Special thanks go to Y. Sato, M. Nakaoka, and R. Ohtafor technical assistance. We also thank M. Seki (Tohoku University)for helpful discussions and M. C. Whitby (University of Oxford)for critical reading and discussion.

Financial support was provided in part by a grant from CoreResearch for Evolutional Science and Technology (CREST) of JapanScience and Technology Corporation; by a Center of Excellence(COE) grant to S.T.; by a Grant-In-Aid for Priority Areas toH.S. for Scientific Research from the Ministry of Education,Culture, Sports, Science and Technology of Japan; and by grantsfrom The Uehara Memorial Foundation and The Naito Foundation.

FOOTNOTES

* Corresponding author. Mailing address: Department of Radiation Genetics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. Phone: 81 75 753 4410. Fax: 81 75 753 4419. E-mail: stakeda{at}rg.med.kyoto-u.ac.jp

Published ahead of print on 5 February 2007.

Supplemental material for this article may be found at http://mcb.asm.org/.

REFERENCES

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