Partial Depletion of Histone H4 Increases Homologous Recombination-Mediated Genetic Instability

DNA replication can be a source of genetic instability. Giventhe tight connection between DNA replication and nucleosomeassembly, we analyzed the effect of a partial depletion of histoneH4 on genetic instability mediated by homologous recombination.A Saccharomyces cerevisiae strain was constructed in which theexpression of histone H4 was driven by the regulated tet promoter.In agreement with defective nucleosome assembly, partial depletionof histone H4 led to subtle changes in plasmid superhelicaldensity and chromatin sensitivity to micrococcal nuclease. Underthese conditions, homologous recombination between ectopic DNAsequences was increased 20-fold above the wild-type levels.This hyperrecombination was not associated with either defectiverepair or transcription but with an accumulation of recombinogenicDNA lesions during the S and G₂/M phases, as determined by anincrease in the proportion of budded cells containing Rad52-yellowfluorescent protein foci. Consistently, partial depletion ofhistone H4 caused a delay during the S and G₂/M phases. Ourresults suggest that histone deposition defects lead to theformation of recombinogenic DNA structures during replicationthat increase genomic instability.

INTRODUCTION

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Introduction

Genetic instability leads to loss of cell fitness and is characteristicof cancer and a number of genetic diseases (31). This instabilitycan be associated with errors during DNA replication. Thus,it is induced by either mutations affecting replication forkprogression or replication inhibitors. In addition, geneticinstability arises in S-phase checkpoint mutants in the absenceof genotoxic agents, suggesting that DNA replication per secan be a source of spontaneous DNA rearrangements (28). Partof this instability involves DNA exchanges between homologoussequences, indicating that chromosomal rearrangements may resultvia homologous recombination from DNA lesions generated duringreplication. In accordance with this, homologous recombinationhas been shown to be required for the rescue of stalled replicationforks in bacteria (13, 36, 50), and recombination intermediateshave been physically detected during defective DNA replicationin Saccharomyces cerevisiae (55, 69).

Newly replicated DNA is rapidly assembled into chromatin ina stepwise manner, with the initial deposition of the histoneH3/H4 tetramers, followed by binding of two histone H2A/H2Bdimers. Nucleosome assembly is catalyzed by histone chaperonesand ATP-dependent chromatin remodeling complexes. Two differentchaperones, conserved from S. cerevisiae to humans, have beeninvolved in replication-dependent chromatin assembly: CAF-1(chromatin assembly factor 1) and Asf1 (antisilencing function1) (1, 37, 59). CAF-1 and Asf1 physically interact with eachother, cooperate in chromatin assembly in vitro, and participatewith redundant and specific roles in heterochromatin silencing,transcription, and DNA repair (29, 51, 60). In addition to CAF-1and Asf1, the histone chaperone Spt6 has been shown to controlchromatin structure in vivo and to participate in nucleosomeassembly in vitro (9).

DNA replication and chromatin assembly are tightly coordinated.The coupling between DNA replication and chromatin assemblyis mediated by the proliferating cell nuclear antigen, whichinteracts physically with the largest subunit of CAF-1 (52)and functionally with both CAF-1 and Asf1 in heterochromatinsilencing and in vitro chromatin assembly (29, 51). In addition,the coordination of DNA replication and nucleosome assemblyrelies on the coregulation of the levels of DNA and histonesynthesis. Thus, histone gene expression is cell cycle regulatedto ensure the accumulation of histones during DNA replication:histone genes are actively transcribed in late G₁/early S andrepressed in early G₁, G₂, and M phases (22, 42, 56). Consistently,replication inhibitors and mutations that block DNA synthesislead to histone gene repression from S. cerevisiae to humancells (34, 54), and repression of histone genes inhibits DNAsynthesis in human cells (40). In addition, a Rad53-dependentsurveillance mechanism that regulates histone levels duringboth normal cell cycle progression and in response to DNA damagein S. cerevisiae has been recently reported (21).

The absence of the Asf1 chromatin assembly factor in S. cerevisiaeincreases genomic instability and sister chromatid exchange,suggesting that defective chromatin assembly may generate recombinogenicDNA lesions during DNA replication (46). Nevertheless, the functionof Asf1 is not restricted to DNA replication. Asf1 is requiredfor histone deposition during nucleotide excision repair invitro (38) and for the repair of double-strand breaks (DSBs)and DNA lesions caused by UV light, methyl-methane sulfonate(MMS), and hydroxyurea (HU) in vivo (30, 60). The activity ofAsf1 in DNA repair seems to be mediated by its dynamic interactionwith the checkpoint protein Rad53 (15, 23). In addition, Asf1participates in the chromatin remodeling required for transcriptionregulation (2, 58).

To determine whether inefficient chromatin assembly during DNAreplication leads to genetic instability mediated by homologousrecombination, a yeast strain expressing histone H4 under thecontrol of the regulated tet promoter was constructed. As expectedfrom the requirement for high histone levels for proper nucleosomeassembly during replication, partial depletion of histone H4altered chromatin structure. Importantly, such a depletion ledto an increase in the frequency of homologous recombinationthat was associated with an accumulation of budded cells containingRad52-yellow fluorescent protein (YFP) foci and with a delayof the cell cycle during the S and G₂/M phases. The increasein recombination was not associated with defective DNA repairor transcription. These results link replication-dependent chromatinassembly and the control of genetic instability mediated byhomologous recombination.

MATERIALS AND METHODS

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Materials and Methods

Yeast strains and growth conditions. Yeast strains used in this study are listed in Table 1. Yeastcells were grown in synthetic complete (SC) medium as describedpreviously (26). Doxycycline (DOX) was added to the medium at0.25 or 5 µg/ml as indicated.

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TABLE 1. S. cerevisiae strains used in this study^a

Plasmids. All plasmids used were centromeric. Plasmids pRS314-GL (44),pRS314-LNA, pRS314-LNAT (47), pRS316-SU, and pRS316-LY ${Delta}$ NS (18)contain the different recombination systems used in this study.p413TARtetH4 contains HHF2 under the control of the tet promoter.p413TARtetH4 was constructed by inserting the following fragmentsinto the vector pRS413 (53): the XhoI-EcoRI fragment from pCM176(8), containing the tetR'::VP16 activator, at the XhoI-EcoRIsite; the SphI (blunt ended)-XhoI fragment from pCM242 (8),containing the tetR-SSN6 repressor, at the ApaI (blunt ended)-XhoIsite; the XhoI (blunt ended)-BamHI fragment from pCM176, containingthe ADHter::tetO7::CYC1TATA sequence, at the NotI (blunt ended)-BamHIsite; and a BamHI fragment containing the HHF2 gene amplifiedby PCR with oligonucleotides 5'-GCATGGATCCCAATAAAATAATGTCCGGTA-3'and 5'-TGAAGGATCCTTAGCCAGTGACTCAAAATT-3', at the BamHI site.pWJ1344 is a centromeric plasmid containing the Rad52-YFP construct(R. Rothstein, Columbia University).

Recombination and DNA repair assays. Spontaneous recombination frequencies in plasmids harboringdirect and inverted repeat systems were obtained by fluctuationassays as the median value of six independent colonies as describedpreviously (45). Yeast colonies were isolated in SC medium containingeither 2% glucose or 2% galactose as indicated (44). MMS andHU sensitivities were determined by 10-fold serial dilutionsplated onto SC medium containing either MMS or HU. Sensitivityto UV light was determined as previously described (20).

Flow cytometry. Analysis of DNA content was performed by flow cytometry (12).Mid-log-phase cells were fixed in 70% ethanol and then incubatedfor 8 h in phosphate-buffered saline with 1 mg of RNase A/mland stained for 30 min with 5 µg of propidium iodide/ml.Samples were sonicated to separate single cells and analyzedin a FACSCalibur flow cytometer (Becton Dickinson). Mid-log-phaseBY4741 and BYtetH4l-1C cell growth with 5 µg of DOX/mlwas arrested in G₁ by twice adding ${alpha}$ factor at 2 and 5 µg/mlat 1- and 2-h intervals, respectively. For synchronization ofBYtetH4l-1C growth with 0.25 µg of DOX/ml, ${alpha}$ factor at5 µg/ml was added four times at intervals of 90 min. Then,cells were washed three times to remove the ${alpha}$ factor and releasedin fresh medium for different times before DNA content analysis.

Chromatin analysis by MNase digestion. Mapping of micrococcal nuclease (MNase) cleavage sites was performedas previously described (11). Briefly, spheroplasts were preparedfrom mid-log-phase cells and then treated with different amountsof MNase. MNase-cleaved genomic DNA was extracted and run in0.8% agarose gels for bulk chromatin analysis. Nucleosome positioningalong the endogenous GAL1 gene and the SU recombination systemwas performed by indirect end labeling: MNase-treated DNA wasrestricted with either EcoRI (GAL1) or ClaI (pRS316-SU), resolvedin a 1.5% agarose gel, blotted onto a Quiabrane Nylon-Plus membrane(Quiagen), and probed with either the 196-bp fragment immediatelydownstream of the EcoRI site (GAL1) or the 221-bp fragment immediatelydownstream of the ClaI site (pRS316-SU).

Plasmid supercoiling analysis. Total DNA from yeast cells transformed with pRS316-SU was isolatedas described by Allers and Lichten (6) with the modificationsof Wellinger et al. (63) in the absence of hexamine cobalt trichloride.DNA samples were run in 0.7% agarose gels containing either4 or 8 µg of chloroquine/ml for 48 h at 2 V/cm to resolveplasmid topoisomers (10). These were probed with either a ClaI-EcoRVLEU2 (pRS316-SU) or an EcoRV-NdeI FLP1 (2µm circle) fragmentand quantified in a Fuji FLA3000 reader.

RNA analysis. Yeast RNA was extracted and analyzed by Northern hybridizationas previously described (47). mRNA was probed with specificDNA fragments, quantified in a Fuji FLA3000 reader, and normalizedwith respect to the 25S rRNA value.

Western blot analysis. Yeast protein extracts were prepared essentially as describedpreviously (16), except that 20% trichloroacetic acid-treatedcells were washed three times with acetone. Protein extractswere run on a sodium dodecyl sulfate-15% polyacrylamide gel,and histone H4 was detected with histone H4 polyclonal antibody(Abcam Ltd.) by standard Western analysis.

Detection of Rad52-YFP. Rad52-YFP foci from mid-log-phase cells transformed with plasmidpWJ1344 were visualized with a Leyca TCS SL confocal microscope(excitation, 514 nm; emission, 535 nm) as described by Lisbyet al. (33).

RESULTS

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Results

Construction of a yeast strain partially depleted of histone H4. In order to understand the role of chromatin assembly in geneticinstability, we constructed a yeast strain in which histoneH4 could be partially depleted (Fig. 1A). This strain (t::HHF2)is a null mutant for the two copies of the histone H4 gene (hhf1 ${Delta}$ hhf2 ${Delta}$ ) and contains a plasmid (p413TARtetH4) expressing HHF2under the control of the bacterial tet promoter. This plasmidharbors copies of the tetR'-SSN6 and tetR-VP16 fusions, whichpositively regulate the tet promoter in response to DOX in adose-dependent manner (8).

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FIG. 1. Construction of a yeast strain (t::HHF2) that can be gradually depleted of histone H4. (A) Scheme of the main genotype of strain t::HHF2. This strain is a null mutant for the two copies of the histone H4 gene (hhf1 ${Delta}$ hhf2 ${Delta}$ ) that contains a plasmid (p413TARtetH4) expressing HHF2 under the control of the bacterial tet promoter, which responds to DOX in a dose-dependent manner. (B) Growth analysis of BYtetH4-10D at 5 (t::HHF2[5]) or 0.25 (t::HHF2[0.25]) µg of DOX/ml or in the absence of DOX and of BY4741 at 5 µg of DOX/ml (wild type [wt]). Cell growth of BY4741 was not affected by the presence or absence of DOX (data not shown). (C) Histone H4 content from strains described in panel B as determined by Western blot analysis. Total protein content was determined by Coomassie staining. The luminescence signals corresponding to histone H4 in t::HHF2[5] and t::HHF2[0.25] cells were 71 and 52% of the wild-type levels, respectively.

Since histone H4 is essential for cell viability, t::HHF2 cellsdo not grow in the absence of DOX (the tet promoter is repressed)and are extremely sick in 0.25 µg of DOX/ml (t::HHF2[0.25]),which leads to a reduction in the level of histone H4 and adoubling time of 5 h versus 2 h for the wild type (Fig. 1B and C).This growth defect was partially rescued with 5 µgof DOX/ml (t::HHF2[5]), which fully activates the tet promoterand results in cells with a slight reduction in the level ofhistone H4 and a doubling time of 3 h (Fig. 1B and C). Therefore,t::HHF2 cells are depleted partially of histone H4, which leadsto growth defects that are more severe as the expression ofhistone H4 from the tet promoter is reduced.

Partial depletion of histone H4 affects chromatin assembly. To analyze the effect of partial depletion of histone H4 onnucleosome assembly, plasmid supercoiling density and chromatinaccessibility to MNase were determined. Since the assembly ofone nucleosome introduces one negative superhelical turn ina circular DNA molecule (62, 65), a loss of plasmid superhelicaldensity in cells partially depleted of histone H4 was expected.As can be seen in Fig. 2A, the endogenous 2µm plasmidin t::HHF2 cells displayed a shift in the distribution of topoisomersthat indicated a loss of negative supercoiling, whereas thecentromeric plasmid pRS314-SU displayed an increase in DNA supercoilingthat was similar to that observed in the absence of the chromatinassembly factor Asf1 (data not shown; 46).

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FIG. 2. Analysis of plasmid superhelical density, chromatin accessibility, and nucleosome positioning in wild-type and t::HHF2 cells. (A) Topoisomer distribution of centromeric pRS316-SU and multicopy 2µm plasmids after electrophoresis in agarose gels containing 4 µg of chloroquine/ml. At this chloroquine concentration, negatively supercoiled topoisomers are resolved, consistent with a slower migration with 8 µg of chloroquine/ml (data not shown). r and sc indicate relaxed and negatively supercoiled plasmids, respectively. (B) MNase accessibility of bulk chromatin. MN, MNase I. (C and D) MNase digestion patterns of the endogenous GAL1 gene and the intervening sequence between the inverted leu2 repeats of the SU repeat system, respectively. A scheme of the analyzed sequences is shown on the left of each panel. Bands whose intensity is modified relative to the wild type are indicated with asterisks. Strains and other details are the same as for Fig. 1B.

Partial MNase digestion of bulk chromatin leads to the formationof a nucleosome ladder (41). As observed in Fig. 2B, this ladderwas more diffuse in t::HHF2[0.25] cells, pointing to a generalloss of nucleosome density. However, the depletion of histoneH4 in t::HHF2[5] cells was not sufficient to induce this chromatinalteration.

For a more detailed molecular analysis of the effect of partialdepletion of histone H4 on chromatin structure, we determinednucleosome positioning along two different DNA sequences: theendogenous GAL1 promoter and the LEU2 promoter of the SU recombinationsystem. Figure 2C shows that nucleosomes remained well positionedalong the regulatory region of GAL1 in both t::HHF2[0.25] andt::HHF2[5] cells. In the case of the SU recombination system,the LEU2 promoter region was more sensitive to MNase in t::HHF2[0.25]cells than in wild-type cells (Fig. 2D). As previously observedfor bulk chromatin, most of these alterations were suppressedin t::HHF2[5] cells. Taken together, our results suggest thatchromatin assembly is affected in t::HHF2 cells, leading tosubtle changes in chromatin structure.

Partial depletion of histone H4 increases homologous recombination. To determine the effect of partial depletion of histone H4 onhomologous recombination, we used intramolecular recombinationsystems based on two direct repeats of a 0.6-kb internal fragmentof the LEU2 gene (Fig. 3A, LY ${Delta}$ NS system). Recombination betweenthese repeats leads to the deletion of the intervening sequenceand one of the repeats, generating a wild-type copy of the LEU2gene (45). The effect of partial depletion of histone H4 oninversions in the recombination system SU was also analyzed;the SU system differs from the LY ${Delta}$ NS system only in the orientationof the repeats (Fig. 3A). Recombination between these repeatsleads to the inversion of the intervening sequence and the formationof a wild-type copy of the LEU2 gene (45). As shown in Fig.3A, deletions and inversions increased 5- to 10-fold above wild-typelevels in hhf1 ${Delta}$ and hhf2 ${Delta}$ single mutants, in which histone H4RNA accumulated at 40 and 80% of the wild-type levels, respectively(data not shown). Notably, the frequency of deletions increased25- and 14-fold in t::HHF2[0.25] and t::HHF2[5] cells, respectively,and the frequency of inversions increased 22- and 18-fold, respectively(Fig. 3A). Therefore, partial depletion of histone H4 causesgenetic instability mediated by homologous recombination.

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FIG. 3. Recombination in cells partially depleted of histone H4. (A) Frequencies of Leu⁺ recombinants in BY4741 (wild type [wt]), Y03144 (hhf1 ${Delta}$ ), and Y15356 (hhf2 ${Delta}$ ) at 5 µg of DOX/ml and BYtetH4-10D at either 5 (t::HHF2[5]) or 0.25 (t::HHF2[0.25]) µg of DOX/ml. (B) Frequencies of Leu⁺ recombinants in BY4741 (wt), BY51 (rad51 ${Delta}$ ), BY59 (rad59 ${Delta}$ ), Y10540 (rad52 ${Delta}$ ), BYtetH4-10D (t::HHF2[5]), BY51tetH4 (t::HHF2[5] rad51 ${Delta}$ ), BY59tetH4 (t::HHF2[5] rad59 ${Delta}$ ), and BY52tetH4 (t::HHF2[5] rad52 ${Delta}$ ) at 5 µg of DOX/ml. All strains carried either the pRS316-LY ${Delta}$ NS or the pRS316-SU plasmid, harboring the direct repeat system LY ${Delta}$ NS or the inverted repeat system SU, respectively. A scheme of each recombination system is shown at the top of the panels. The frequencies of Leu⁺ recombinants of wild-type, hhf1 ${Delta}$ , hhf2 ${Delta}$ , rad51 ${Delta}$ , rad52 ${Delta}$ , and rad59 ${Delta}$ strains were not affected by the presence or absence of DOX (data not shown). The averages and standard deviations of three or four median frequencies obtained with two or three independent transformants are shown.

To determine the genetic basis of the recombination events inducedby partial depletion of histone H4, we analyzed the effectsof rad52 ${Delta}$ , rad59 ${Delta}$ , and rad51 ${Delta}$ mutations (Fig. 3B), whose geneticconsequences on recombination are known to depend on the typeof event. Thus, deletions and inversions require Rad52 and Rad59,but they can occur efficiently in the absence of Rad51 (5, 7,24, 48). As observed with wild-type cells, both deletions andinversions were partially dependent on Rad59 and completelydependent on Rad52 in t::HHF2 cells. However, the level of inversionsstimulated by histone H4 depletion in rad51 ${Delta}$ cells was fourfoldabove the level in RAD51 cells. This indicates that homologousrecombination in the absence of Rad51 is more efficient underconditions of histone H4 depletion. This is in agreement withthe increase in recombination in rad51 ${Delta}$ cells carrying eithera single copy of the histone H2A and H2B loci or the spt6-140mutation (35) and the requirement for Rad51 in DSB-induced recombinationin the silenced mating-type loci (57).

Our results suggest that recombinational repair may be a highlyrelevant process for the viability of cells partially depletedof histone H4. Consistent with this conclusion rad52 ${Delta}$ t::HHF2cells grew poorly in 5 µg of DOX/ml and were extremelysick in 0.25 µg of DOX/ml (Fig. 4A).

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FIG. 4. DNA repair in cells partially depleted of histone H4. (A) Growth analysis of BYtetH4-10D (t::HHF2) and BY52tetH4 (t::HHF2 rad52 ${Delta}$ ) at either 5 (t::HHF2[5]) or 0.25 (t::HHF2[0.25]) µg of DOX/ml. (B, C, and D) UV light, HU, and MMS sensitivities of the strains indicated in the legend to Fig. 1, respectively. rad1 ${Delta}$ , asf1 ${Delta}$ , and rad52 ${Delta}$ strains are controls for UV light-, HU-, and MMS-induced DNA repair, respectively, and were analyzed at both 5 (t::HHF2[5]) and 0.25 (t::HHF2[0.25]) µg of DOX/ml. Since UV light, HU, and MMS sensitivities of wild-type, rad52 ${Delta}$ , asf1 ${Delta}$ , and rad1 ${Delta}$ strains were not affected by the presence or absence of DOX (data not shown), only the data for 5 µg of DOX/ml are shown for these strains.

Hyperrecombination induced by partial depletion of histone H4 is not associated with defective repair or transcription. Since the absence of the Asf1 and CAF1 chromatin assembly factorsleads to defects in DNA repair and transcription (2, 17, 30,58, 60), it was important to know whether partial depletionof histone H4 caused similar defects that could be associatedwith the increase in recombination. Thus, we analyzed the capacityof t::HHF2 cells to repair DNA lesions induced by either thealkylating agent MMS, the replication inhibitor HU, or UV light,which are subject to different DNA repair mechanisms (64). Ascan be seen in Fig. 4, t::HHF2 cell viability was not reducedby MMS or UV light and showed only a slight decrease at highlevels of HU, as determined by CFU. Therefore, hyperrecombinationin t::HHF2 cells seems not to be due to the recombinationalprocessing of unrepaired DNA lesions.

Histone H4 depletion affects global transcription regulationbut not the expression levels of recombination proteins (66).However, since transcription induces homologous recombination(4), we wondered whether recombination induced by histone H4depletion could be mediated by transcription. To analyze thispossibility, three recombination systems based on the same directrepeats as those in the LY ${Delta}$ NS system were used (Fig. 5). Thefirst two systems differ in the presence (LNAT) or absence (LNA)of a terminator just downstream of the first leu2 repeat, whichimpedes transcription elongation progress through the interveningsequence located between the repeats. The third system (GL)is under the control of the GAL1 promoter; thus, transcriptioncould be turned on and off with galactose or glucose, respectively.Recombination was analyzed in t::HHF2[5] cells for the LNA andLNAT systems because recombination levels were similar at 5and 0.25 µg of DOX/ml (Fig. 3) and in hhf2 ${Delta}$ cells for theGL system because the GAL1 promoter was derepressed in t::HHF2cells (data not shown). As can be seen in Fig. 5, hyperrecombinationin t::HHF2[5] and hhf2 ${Delta}$ cells was independent of the transcriptionalstate of the DNA sequence (compare the LNA and GL [galactose]systems versus the LNAT and GL [glucose] systems). Therefore,hyperrecombination mediated by partial depletion of histoneH4 is not dependent on transcription.

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FIG. 5. Effect of transcription in recombination induced by partial depletion of histone H4. (A) leu2 RNA levels and frequencies of Leu⁺ recombinants of strains Y07202 (wild type [wt]) and BYtetH4-10D (t::HHF2[5]) transformed with pRS314-LNA or pRS314-LNAT and grown in 5 µg of DOX/ml. (B) leu2 RNA levels and frequencies of Leu⁺ recombinants of strains Y07202 (wt) and BYth2-1B (hhf2 ${Delta}$ ) transformed with pRS314-GL and grown in either glucose- or galactose-containing medium, in which the GAL1 promoter of the GL system is either repressed or activated, respectively. Schemes of the direct repeat systems are shown. Dashed arrows indicate the transcripts produced by the recombination systems. As a probe, the ClaI-EcoRV LEU2 fragment was used. The averages and standard deviations of two to four median frequencies obtained with two to four independent transformants are shown.

Hyperrecombination induced by partial depletion of histone H4 is related to defective DNA replication. Given the tight connection between nucleosome assembly and DNAsynthesis, we wondered whether hyperrecombination in t::HHF2cells was linked to DNA replication defects. Thus, we wouldexpect that growth retardation in t::HHF2 cells was due to adelay during the S and G₂/M phases of the cell cycle. In accordancewith this, DNA content analysis of asynchronous cultures byflow cytometry showed an accumulation of t::HHF2 cells duringthe S and G₂/M phases with both 0.25 and 5 µg of DOX/ml(Fig. 6A). Also, t::HHF2 cells accumulated as budded cells andshowed a heterogeneous increase in cell size (Fig. 6A). To confirmthe delay during S phase, cell cycle progression of ${alpha}$ -factor-synchronizedcultures was monitored by flow cytometry. As shown in Fig. 6B(time zero), a fraction of t::HHF2 cells could not be arrestedin G₁ with ${alpha}$ factor, particularly in 0.25 µg of DOX/ml.It is possible that this fraction corresponds to those t::HHF2cells accumulated in G₂/M that do not to progress into G₁ andconsequently do not respond to ${alpha}$ factor. Alternatively, t::HHF2cells could be less sensitive to ${alpha}$ factor. Indeed, t::HHF2[0.25]cells required five times more ${alpha}$ factor for synchronization thanwild-type cells. This reduced sensitivity could also explainthe apparent early entrance of t::HHF2[0.25] cells into S phaseupon ${alpha}$ -factor release (Fig. 6B, time 15'). Nevertheless, it isclear that most wild-type cells reached G₂ while t::HHF2 cellsaccumulated in S phase at 30 min upon release from ${alpha}$ -factor arrest.Therefore, cells partially depleted of histone H4 accumulatein S and G₂/M phases, in agreement with replication-associatedcell cycle defects.

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FIG. 6. Cell cycle analysis of cells partially depleted of histone H4. (A) DNA content and cell morphology of asynchronous cultures of the strains and at the conditions indicated in the legend to Fig. 1. Similar profiles were obtained with BYtetH4l-1C (data not shown). (B) Cell cycle progression of BYtetH4l-1C at either 5 (t::HHF2[5]) or 0.25 (t::HHF2[0.25]) µg of DOX/ml and of BY4741 at 5 µg of DOX/ml (wild type [wt]). DNA content of wild-type and t::HHF2 cells was determined by flow cytometry at the indicated times upon release from ${alpha}$ -factor arrest in G₁.

To determine the importance of DNA replication, recombinationin t::HHF2 cells was measured in the presence of HU, an inhibitorof nucleotide reductase, which pauses replication by limitingdeoxynucleoside triphosphate pools (68). HU-mediated impairmentof DNA replication has been shown to lead to single- and double-strandDNA breaks and an increase in homologous recombination (19,49). Importantly, HU-induced DNA damage is repaired in t::HHF2cells (Fig. 4C). As shown in Fig. 7A, HU treatment increasedrecombination up to 15-fold in wild-type cells and only 2-foldin t::HHF2[5] cells. In addition, recombination frequencieswere the same in wild-type and t::HHF2[5] cells at 100 mM HU.The lack of an additive effect of histone H4 depletion and HUon recombination is consistent with the idea that replicationis the major process affected in histone H4-depleted cells.In this regard, the spt16-197 point mutation of SPT16, an essentialchromatin remodeling gene involved in replication (25), doesnot affect growth of t::HHF2 cells at either 5 or 0.25 µgof DOX/ml (data not shown). These results are consistent withthe idea that the recombination events induced by partial depletionof histone H4 may by associated with replication defects.

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FIG. 7. (A) Effect of HU on recombination induced by partial depletion of histone H4. Frequencies of Leu⁺ recombinants of the strains indicated in the legend to Fig. 1 grown with 5 µg of DOX/ml and either 0, 25, or 100 mM HU. (B) Accumulation of Rad52-YFP foci in t::HHF2 cells. Rad52-YFP foci were visualized by fluorescence microscopy in asynchronous cultures of BYtetH4-10D transformed with pWJ1344 and grown in either 5 (t::HHF2[5]) or 0.25 (t::HHF2[0.25]) µg of DOX/ml and of BY4741 transformed with pWJ1344 and grown in 5 µg of DOX/ml (wild type [wt]). The percentages of unbudded (G₁) and budded (S and G₂/M) wild-type and t::HHF2 cells containing Rad52-YFP foci are shown. The total numbers of analyzed cells were 50 for unbudded cells and 150 for budded cells. The averages and standard deviations of two independent experiments are plotted.

Partial depletion of histone H4 leads to an accumulation of Rad52 foci during DNA replication. To determine whether recombinogenic DNA lesions were accumulatedduring replication as a consequence of the depletion of histoneH4, the formation of Rad52-YFP foci was analyzed. These focihave previously been shown to appear both spontaneously andin response to DSBs during S and G₂/M phases, providing a specificlink between DNA replication and homologous recombination (32,33). As previously published, Rad52-YFP foci were detected in3% of unbudded (G₁) and 18% of budded (S and G₂/M) wild-typecells (Fig. 7B) (33). In t::HHF2 cells the Rad52-YFP foci appearedat wild-type levels (4%) in unbudded cells but increased to46 and 51% in budded cells in the presence of 5 and 0.25 µgof DOX/ml, respectively (Fig. 7B). Therefore, partial depletionof histone H4 leads to an accumulation of DNA lesions duringDNA replication that are repaired by homologous recombination.

DISCUSSION

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Discussion

In this study we show that partial depletion of histone H4 affectschromatin assembly and causes genetic instability by increasingthe frequency of homologous recombination between ectopic DNAsequences. This increase in recombination is associated withan accumulation of Rad52-YFP foci and a cell cycle delay duringthe S and G₂/M phases. Our results suggest that defective chromatinassembly mediated by partial depletion of histone H4 impairsDNA synthesis and leads to the formation of recombinogenic structures.

To impair nucleosome deposition during DNA replication, a yeaststrain (t::HHF2) in which the expression of histone H4 is underthe control of the regulated tet promoter was constructed. Thisstrain can be gradually depleted of histone H4. Our resultssuggest that chromatin assembly is affected in this strain.Thus, partial depletion of histone H4 leads to subtle changesin plasmid superhelical density and chromatin accessibilityto MNase, particularly at 0.25 µg of DOX/ml. Also, t::HHF2cells are delayed in the S and G₂/M phases and accumulate asbudded cells with a heterogeneous increase in cell size. Ourresults are reminiscent of those obtained for cells completelydepleted of histone H4, which show dramatic changes in plasmidtopology and chromatin accessibility and stop growing at theS and G₂/M phases of the cell cycle (27). It is worth notingthat chromatin assembly factor mutants display phenotypes similarto those of t::HHF2 cells. Thus, yeast mutants lacking the chromatinassembly factor Asf1 accumulate as large cells in S and G₂/Mphases (60) and display similar alterations in chromatin accessibilityto MNase (46). In addition, the superhelical density of thecentromeric plasmid pRS316-SU is increased in t::HHF2 and asf1 ${Delta}$ cells (Fig. 2A; 46), whereas the supercoiling of the endogenous2µm plasmid is decreased in t::HHF2 cells (Fig. 2A) andincreased in asf1 ${Delta}$ cells (data not shown; 3). Similar observationshave been reported for human cells, in which HIRA-mediated histonerepression and expression of a dominant-negative mutation ofthe chromatin assembly factor p150CAF-1 cause chromatin hypersensitivityand replication defects (40, 67). In this sense, it is worthnoting that histone depletion causes S-phase arrest in humancells (40) but S-phase delay and G₂/M arrest in yeast cellsas determined by flow cytometry (Fig. 6B; 27).

The most relevant observation of this study is that partialdepletion of histone H4 increases recombination between ectopicDNA sequences up to 20-fold above wild-type levels. These recombinationevents are RAD52 dependent, as expected for events occurringby homologous recombination mechanisms. The importance of recombinationalrepair in cells depleted partially of histone H4 is supportedby the severe growth defects of t::HHF2 rad52 ${Delta}$ cells. Notably,recombination was 5- to 10-fold above wild-type levels in hhf1 ${Delta}$ and hhf2 ${Delta}$ single mutants, in which histone H4 RNA levels werereduced to 80% of that of the wild type. Our results stronglysuggest that DNA stability is sensitive to slight reductionsin the pool of histone H4. In accordance with this idea, deletionof HHF1 has been shown to result in telomeric sequence instability,manifested by changes in their length and sequence organization(61). The effect on recombination is less evident for a deletionof one of the two genes coding for histones H2A and H2B (35).Altogether, these results are consistent with the central roleof the H3/H4 tetramer in nucleosome assembly (59).

The increase in recombination and its association with an accumulationof Rad52-YFP foci in t::HHF2 cells are consistent with a generalrole of proper chromatin assembly in preventing genetic instability.This is strongly supported by our recent observation that yeastcells lacking the chromatin assembly factor Asf1 also displayan increase in recombination and an accumulation of Rad52-YFPfoci (46). In contrast to cells lacking Asf1, histone H4 depletiondoes not impair DNA repair, and consequently, this rules outthe possibility that the increase in recombination could bedue to the channeling of unrepaired DNA lesions into homologousrecombination. The influence of chromatin assembly in genomeintegrity is also supported by the observations that a thermosensitivespt6-140 mutation leads to hyperrecombination (35) and thata chromatin assembly factor p150CAF-1 dominant-negative mutantaccumulates DNA breaks and induces S-phase arrest in human cells(67). In the same vein, the absence of the yeast chromatin assemblyfactors CAF-1 and Asf1 leads to gross chromosomal rearrangements(GCRs) (39). These rearrangements are Rad52 and homology independent.Indeed, the frequency of GCRs is synergistically increased inrad52 ${Delta}$ mutants, indicating that homologous recombination partiallysuppresses GCRs (39). The results of the present study provideevidence that the defects in chromatin assembly also increasegenetic instability mediated by homologous recombination.

The tight connection between chromatin assembly and DNA synthesisprovides a framework to explain the genetic instability reportedhere. Defective chromatin assembly could impair DNA synthesis,thus leading to the accumulation of recombinogenic structures.Consistent with this interpretation, we have shown that t::HHF2cells are delayed during S phase and accumulate in G₂/M. Also,the observation that hyperrecombination in t::HHF2 cells isnot further increased by the presence of the replication inhibitorHU suggests that it results from lesions generated during DNAreplication. Indeed, t::HHF2 cells accumulate Rad52-YFP foci(Fig. 7), which have been shown to appear spontaneously andin response to DSBs during the S and G₂/M phases (33). Our resultsare in accordance with the observation that the lethality associatedwith total depletion of histone H4 occurs in S phase (27). Altogether,these results strengthen the idea that homologous recombinationhas an important role in the control of the accuracy of DNAreplication. Presumably, defective nucleosome assembly increasesthe basal rate of replication-mediated DNA lesions and, in turn,the requirement of the recombination machinery for the repairof these lesions. Partial depletion of histone H4 provides aunique tool to study the role of proper chromatin assembly ingenome integrity during replication.

We do not yet know the nature of the recombinogenic structuresgenerated in t::HHF2 cells. DSBs have been extensively shownto induce homologous recombination (43), and it has recentlybeen reported that phosphorylation of histone H2A (P-H2A), whichoccurs in response to DSBs (14), increases in yeast asf1 ${Delta}$ mutants(46) and in human cells defective in the chromatin assemblyfactor p150CAF-1 (67). We have not been able to detect P-H2Ain t::HHF2 cells (data not shown). Whether DSBs, replicationfork blocks, or single-stranded DNA gaps or tails are the primarystructures leading to recombination as a consequence of impairednucleosome assembly remains to be determined.

ACKNOWLEDGMENTS

We thank R. Rothstein for providing the Rad52-YFP construct,G. Vicent for the anti-H4 antibodies, M. Nieto for excellenttechnical assistance, M. Fidalgo for help with confocal microscopyand flow cytometry, R. E. Wellinger and M. C. Muñoz-Centenofor critical reading of the manuscript, and Diane Haun for stylesupervision.

The research was funded by the Spanish Ministry of Science andTechnology (BMC2000-0439 and SAF2003-00204 grants).

FOOTNOTES

* Corresponding author. Mailing address: Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Avda. Reina Mercedes 6, 41012 Seville, Spain. Phone: (34) 954557107. Fax: (34) 954557104. E-mail: aguilo{at}us.es.

REFERENCES

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