Rad51-Independent Interchromosomal Double-Strand Break Repair by Gene Conversion Requires Rad52 but Not Rad55, Rad57, or Dmc1

Homologous recombination (HR) is critical for DNA double-strandbreak (DSB) repair and genome stabilization. In yeast, HR iscatalyzed by the Rad51 strand transferase and its "mediators,"including the Rad52 single-strand DNA-annealing protein, twoRad51 paralogs (Rad55 and Rad57), and Rad54. A Rad51 homolog,Dmc1, is important for meiotic HR. In wild-type cells, mostDSB repair results in gene conversion, a conservative HR outcome.Because Rad51 plays a central role in the homology search andstrand invasion steps, DSBs either are not repaired or are repairedby nonconservative single-strand annealing or break-inducedreplication mechanisms in rad51 ${Delta}$ mutants. Although DSB repairby gene conversion in the absence of Rad51 has been reportedfor ectopic HR events (e.g., inverted repeats or between plasmids),Rad51 has been thought to be essential for DSB repair by conservativeinterchromosomal (allelic) gene conversion. Here, we demonstratethat DSBs stimulate gene conversion between homologous chromosomes(allelic conversion) by >30-fold in a rad51 ${Delta}$ mutant. We showthat Rad51-independent allelic conversion and break-inducedreplication occur independently of Rad55, Rad57, and Dmc1 butrequire Rad52. Unlike DSB-induced events, spontaneous allelicconversion was detected in both rad51 ${Delta}$ and rad52 ${Delta}$ mutants, butnot in a rad51 ${Delta}$ rad52 ${Delta}$ double mutant. The frequencies of crossoversassociated with DSB-induced gene conversion were similar inthe wild type and the rad51 ${Delta}$ mutant, but discontinuous conversiontracts were fivefold more frequent and tract lengths were morewidely distributed in the rad51 ${Delta}$ mutant, indicating that heteroduplexDNA has an altered structure, or is processed differently, inthe absence of Rad51.

INTRODUCTION

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INTRODUCTION

DNA repair is critical for genome stability and tumor suppressionin higher eukaryotes. DNA double-strand breaks (DSBs) are criticallesions that result from exposure to exogenous agents (ionizingradiation or genotoxic chemicals) and from endogenous sources,such as nucleases and spontaneous DNA lesions that cause replicationfork collapse. DSBs can be repaired by homologous recombination(HR) or by nonhomologous end joining (NHEJ). In the yeast Saccharomycescerevisiae, the primary DSB repair pathway is homologous recombination(HR), and the key HR proteins are members of the Rad52 epistasisgroup (Rad51, Rad52, Rad54, Rad55, Rad57, and Rad59). The Mre11/Rad50/Xrs2(MRX) complex has roles in both HR and NHEJ. The most commonHR outcome is gene conversion, a conservative and highly accuratemode of DSB repair that occurs with or without an associatedcrossover. In cells with mild to severe HR defects, other outcomesmay occur, including chromosome loss or nonconservative repairby either break-induced replication (BIR) or single-strand annealing(SSA) (10, 16, 25, 27). Inefficient or inaccurate DSB repairby either HR or NHEJ has been linked to the development andprogression of cancer (28, 59).

DSB repair by HR involves a series of steps that begins with5'-to-3' single-strand resection regulated by MRX, and bindingof the single-stranded DNA (ssDNA) tail by replication proteinA. Replication protein A is then exchanged with Rad51 to produceRad51 nucleoprotein filaments that are capable of searchingfor, and invading, homologous sequences elsewhere in the genome(sister chromatids, homologous chromosomes, or repeated sequences).The Rad51-related protein Dmc1 is also required for meioticHR (32). Rad51 filament formation is promoted by the "mediator"proteins Rad52, Rad55, and Rad57; Rad54 also mediates Rad51filament formation in vitro, although filaments form normallyin rad54 ${Delta}$ mutants in vivo (19, 51, 54, 60). Genetic and biochemicalstudies suggest that Rad54 promotes invasion of the intact (donor)duplex by the Rad51 filament and branch migration of the jointmolecule producing heteroduplex DNA (hDNA) and dissociates Rad51from ssDNA to allow repair synthesis by extension of the invading3' end (4, 15, 40, 47, 48). The invading 3' end is extendedby repair synthesis past the site of the DSB and can then dissociatefrom the donor duplex and anneal with the other ssDNA tail ina process termed synthesis-dependent strand annealing, a noncrossoverpathway. Alternatively, both ends may invade a donor, producingHolliday junction intermediates that can be resolved with orwithout crossovers. Mismatches arise in hDNA when interactingduplexes have sequence differences ("markers"). Mismatch repairis typically biased, with the strand from the donor (unbroken)duplex used as a mismatch repair template, resulting in regionsof marker loss in the recipient (broken) duplex that are termedconversion tracts (38). Because most mismatch repair proceedsby a long-patch, excision-based mechanism, conversion tractsare usually continuous, i.e., when two markers flanking a centralmarker are converted, the central marker almost always coconverts.Thus, conversion tracts result from mismatch repair of hDNAproduced during strand invasion and branch migration and whenan extended 3' end anneals to ssDNA during synthesis-dependentstrand annealing.

The Rad51 filament plays a central role in strand invasion,and DSB repair via gene conversion is severely reduced or eliminatedin the rad51 ${Delta}$ mutant, with repair instead occurring by SSA orBIR. With only a single DSB, SSA is limited to interactionsbetween linked, direct repeats as resection to ssDNA tails exposescomplementary single strands that anneal to repair the break.SSA may be important for repair when two or more DSBs occurnear each other (49). Because SSA does not require strand invasion,it is independent of Rad51, Rad54, Rad55, and Rad57 but requiresthe strand-annealing activity of Rad52 (10, 27, 49). BIR wasinitially described in rad51 ${Delta}$ cells (25, 46). Several studieshave revealed an inefficient Rad51-independent BIR mechanismthat requires Rad59, Tid1, and MRX and a more efficient Rad51-dependentmechanism that requires Rad52, Rad55, Rad57, Rad54, and Ku (6,16, 26, 46). Even the more efficient BIR mechanism is rare orabsent in wild-type cells when gene conversion is possible (25,35, 46), although BIR appears to be important for telomerase-independenttelomere maintenance (24).

Despite the central role of Rad51 in strand invasion, Rad51-independentgene conversion has been detected in certain contexts. Spontaneousgene conversion between inverted repeats is reduced only 4-foldby deletion of RAD51, whereas deletion of RAD52 reduces theseevents by 3,000-fold (42). Ectopic gene conversion involvingrepair of plasmid double-strand gaps with plasmid or chromosomalrepair templates was readily detected in the rad51 ${Delta}$ mutant, albeit $~$ 100-fold less efficiently than in the wild type; these recombinationevents were dependent on Rad52 and Rad59 (2). Yeast mating-typeswitching involves intrachromosomal gene conversion resultingfrom repair of an HO nuclease-induced DSB at the MAT locus.This is a lethal event in the rad51 ${Delta}$ mutant; however, MAT switchingdoes occur in the rad51 ${Delta}$ mutant when interacting alleles arepresent on plasmids. These plasmid events were originally explainedas reflecting a relaxed requirement for Rad51 when donor sequencesare not silenced in heterochromatin, as in normal MAT switching(50), but an alternative explanation is that the plasmid eventsdepend on BIR between closely linked alleles (2). Thus, Rad51-independentDSB repair by gene conversion has been detected only for ectopicevents (inverted repeats, or plasmid-chromosome or plasmid-plasmidinteractions); there are no prior reports of such repair betweenchromosomal (allelic) loci in the absence of Rad51 (17).

By using a sensitive DSB repair assay, we demonstrate that yeastis capable of Rad51-independent DSB repair resulting in interchromosomalgene conversion. The efficiency of this repair is similar tothat of Rad51-independent BIR, and these processes share similargenetic requirements: both are independent of Rad55, Rad57,and Dmc1 but require Rad52. Spontaneous gene conversion, however,was detected in the absence of Rad51 or Rad52, but not bothproteins. Crossovers associated with conversion occur at aboutthe same frequency during Rad51-dependent and -independent events,but Rad51-independent gene conversion tracts were frequentlydiscontinuous and generally longer than in the wild type, suggestingthat hDNA forms or is processed differently in the absence ofRad51.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Plasmids and yeast strains. Plasmid pHSS19Trp1rad51 has 849-bp and 1,151-bp fragments fromup- and downstream of RAD51 flanking TRP1. Standard procedureswere used for yeast culture and chromosome modification (35),and the structures of all modified chromosomes were confirmedby Southern and PCR analyses. All yeast strains were isogenicwith diploid JC3598 (37) (MATa-inc/MAT ${alpha}$ -inc ade2-101/ade2-101his3-200:HIS3:telV/his3-200 lys2-801::pHSSGALHO::LYS2/lys2-801trp1- ${Delta}$ 1/trp1- ${Delta}$ 1 leu2- ${Delta}$ 1/leu2- ${Delta}$ 1 RscRI-ura3R-HO432-LEU2/RscBam-ura3-X764-LEU2).GALHO indicates the source of the HO nuclease, which cleavesthe HO site at position 432 in ura3 (HO432). All strain genotypesare listed in Table S1 in the supplemental material. RAD51 wasreplaced with TRP1 by one-step transplacement using pHSS19Trp1rad51.RAD52, RAD55, RAD57, and DMC1 were replaced with KanMX by one-steptransplacement with PCR-amplified fragments from appropriatestrains from the yeast deletion set. Double and triple mutantswere constructed by one-step KanMX replacements or mating/sporulationof appropriate single/double mutants. All markers in and aroundura3, and the telomere-proximal HIS3:telV marker, were confirmedin all strains by mapping PCR amplification products.

Selective and nonselective HR assays. HR was analyzed in diploid yeast strains carrying ura3 heteroallelesat the normal chromosome V locus flanked by pUC19 and LEU2 (35).One copy of ura3 ("recipient") was inactivated by an insertionof a 24-bp HO nuclease site flanked by 11 phenotypically silentmutations that create and/or destroy restriction sites (restrictionfragment length polymorphism markers). These markers were usedto map conversion tracts in recombinant products. This chromosomealso carried a telomere-proximal HIS3 gene 110 kbp upstreamfrom ura3. The second ura3 ("donor") was inactivated by a frameshiftmutation termed X764. An integrated HO gene was regulated bythe GAL1 promoter (GALHO). Cells were incubated in rich mediumwith 2% glycerol for 24 h to deplete intracellular glucose priorto incubation for 6 h in rich medium with 2% galactose (YPGal),or 2% glucose (YPD) as an uninduced control. The depletion ofglucose ensures rapid induction of GALHO in YPGal, which leadsto a single DSB in the recipient ura3 allele. Both MAT locihad single-base mutations, rendering them insensitive to cleavageby HO. Recombinants with short conversion tracts that extendedfrom the HO site past X764 were Ura positive (Ura⁺), while thosewith tracts that included X764 remained Ura negative (Ura^–).In HR-proficient cells, both types of products could be analyzedby a nonselective assay in which cells incubated for 6 h inliquid YPGal or YPD were seeded onto nonselective (YPD) platesand the resulting colonies were identified as Ura⁺ or Ura^–recombinants, or parental Ura^–, using appropriate selectivemedia. Parental colonies were identified as "reinducible" toUra⁺ when transferred first to YPGal plates and then to uracilomission plates (35), whereas Ura^– recombinants were homozygousfor the X764 frameshift mutation and could not be reinducedto Ura⁺. In cells with severe HR deficiencies (e.g., rad51 ${Delta}$ ),only Ura⁺ recombinants were analyzed by using a selective assayin which $~$ 10⁷ cells from YPD or YPGal cultures were directlyplated on uracil omission medium. In either assay, recombinantswere also screened for the His phenotype by replica platingthem onto histidine omission medium. In the nonselective assay,HR frequencies were calculated as the number of Ura⁺ or Ura^–recombinants per YPD colony scored, with 1,000 to 1,500 coloniesscored in each of three or four independent determinations perstrain. In the selective assay, HR frequencies were calculatedas the number of Ura⁺ recombinants per viable cell plated onuracil omission medium, with three or four independent determinationsper strain. The number of viable cells was determined by parallelplating of appropriate dilutions on YPD.

Products isolated in nonselective assays may include those arisingby HR, BIR, chromosome loss, and half-crossovers (25, 35). TheHIS3:telV marker on broken chromosomes was assayed by quantitativereal-time PCR to determine chromosome loss among Ura^–His^– products, as described previously (21). His^–products can result from crossovers in G₂ cells, and these havean associated product with two copies of HIS3:telV (His⁺⁺),identified by PCR. His^– products arising by BIR or half-crossoverscomprise nonloss/noncrossover His^– products (35). BIRyields two chromosomes, and half-crossovers yield only one,so distinguishing these requires quantitative analysis of thechromosome number, but this was not performed because we focusedon His⁺ (gene conversion) products. Gene conversion tract spectrawere generated by rescuing and mapping donor and recipient allelesfrom yeast genomic DNA linked to pUC19, as described previously(22, 35). All rad51 ${Delta}$ products were isolated from independentparent cultures. The rad51 ${Delta}$ rad55 ${Delta}$ rad57 ${Delta}$ products were isolatedfrom a limited set of parent cultures, and two pairs of productswith identical discontinuous tracts were potential sibling pairs,but only one of each pair was counted to ensure product independence.Limited marker analysis of rad52 ${Delta}$ HR products was performed bymapping PCR products amplified from yeast genomic DNA from subclonedproducts; subcloning prevents sectored colonies from being scoredas heterozygous. In this analysis, loss of heterozygosity (LOH)can be detected but linkage information is unavailable becausethe two alleles coamplify. Statistics were calculated by usingt tests unless otherwise specified.

RESULTS

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RESULTS

Rad51-independent DSB repair by allelic gene conversion. HR in the absence of Rad51 has been detected for spontaneousevents and DSB-induced ectopic events (2, 42, 50), but not DSB-inducedallelic (interchromosomal) gene conversions (17, 25, 50). Whiletesting the effects of Rad51 ATPase defects on the repair ofHO nuclease-induced DSBs by allelic gene conversion betweenura3 heteroalleles in diploid yeast (Fig. 1), we constructeda rad51 ${Delta}$ strain as a control. In a nonselective assay that providedestimates of HR, BIR, and chromosome loss frequencies, HR wasnot detected in the rad51 ${Delta}$ mutant. In wild-type cells, nearlyall DSBs in ura3 were repaired by HR (gene conversion); BIRwas not detected, and chromosome loss was very rare (Fig. 2A)(35). In the rad51 ${Delta}$ mutant, HR was replaced by chromosome lossand BIR (Fig. 2A).

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FIG. 1. DSB fates in diploid yeast. Two copies of chromosome V are shown with ura3 alleles inactivated by an HO site insertion or an X764 frameshift (black bars). The chromosome with the HO site has the telomere-proximal HIS3 marker $~$ 100 kbp upstream of ura3. Gene conversion (GC) produces short- and long-tract products. Half of G₂ crossovers result in HIS3 LOH (His^– or His⁺⁺ products), and the other half retain HIS3 heterozygosity. BIR produces only His^– products (above the dashed line). His^– products that are also Ura^– also arise by chromosome loss. Other rare outcomes are listed but not diagrammed. All outcomes were scored in the nonselective assay, but only Ura⁺ products were detected in the selective assay.

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FIG. 2. HR, BIR, and chromosome loss in the wild type (WT) and the rad51 ${Delta}$ mutant. (A) Frequencies of DSB-induced HR, BIR, and chromosome loss were measured with the nonselective assay (GALHO induced). HR includes short- and long-tract gene conversion products (Ura⁺ and Ura^–) with or without associated crossovers (His^– or His⁺), but not His^– BIR products. Chromosome loss was estimated from the fraction of Ura^– His^– products that retained only the donor chromosome. ND, not detected. The values are averages plus SD for four determinations per strain. (B) Average frequencies (plus SD) of spontaneous (GALHO not induced) and DSB-induced Ura⁺ His⁺ gene conversions for four determinations. **, P < 0.0001 for spontaneous versus DSB-induced frequencies.

When rad51 ${Delta}$ colonies were transferred from YPD to YPGal plates(which induces GALHO), incubated for 1 to 2 days, and then transferredto uracil omission medium, many colonies showed Ura⁺ papillae.Such Ura⁺ products could arise by BIR, which is known to occurin the absence of Rad51 (14, 25, 46). However, BIR producesonly His^– products (Fig. 1), yet a substantial fraction( $~$ 50%) of the Ura⁺ papillae were His⁺ (data not shown) and potentiallyarose by allelic gene conversion. Because Ura⁺ papillae arosefar less frequently when colonies were maintained on YPD (withGALHO repressed) prior to transfer to uracil omission medium,the Ura⁺ His⁺ colonies were apparently products of DSB repair.Because these products arose at low frequencies, we employeda sensitive, selective assay to measure Ura⁺ His⁺ inductionlevels in the rad51 ${Delta}$ mutant and in a wild-type control strain.Direct selection of Ura⁺ products detects only short-tract products;long-tract gene conversion products remain Ura^– and arenot detected. The selective assay also fails to detect chromosomeloss and some BIR events (see Materials and Methods). As shownin Fig. 2B, GALHO induction increased the frequency of Ura⁺His⁺ products by 190-fold above spontaneous (uninduced) levelsin wild-type cells. The induced Ura⁺ His⁺ frequency in the rad51 ${Delta}$ mutant was far lower than in the wild type (6.6 x 10^–6versus 9.5 x 10^–2), but spontaneous levels were also reduced,and there was a 33-fold increase in Ura⁺ His⁺ products uponGALHO induction.

Although the absolute frequency of induced Ura⁺ His⁺ productsin the rad51 ${Delta}$ mutant was low, it was unlikely that these productsarose by imprecise NHEJ, because this occurs at very low frequenciesin haploids and even lower frequencies in diploids (5, 29).To rule out NHEJ as a source of the induced Ura⁺ His⁺ products,we performed several control experiments. First, we inducedGALHO in the haploid parent of our wild-type diploid carryingura3 with the HO site (strain JC3441) and a rad51 ${Delta}$ derivative(strain JC3702), but no Ura⁺ products were detected in eitherstrain (<2.5 x 10^–8). Next, we induced GALHO in a diploidthat carried the ura3-HO site allele but lacked a ura3 donorin the homologous chromosome V (strain TP3826); again, no Ura⁺products were detected (<2.5 x 10^–8). Finally, we usedPCR to amplify the ura3 loci from several Ura⁺ His⁺ productsof the original rad51 ${Delta}$ diploid and mapped these products withNcoI (the natural restriction site in the donor opposite theHO site insertion). In all cases, the products were homozygousNcoI⁺, consistent with nonreciprocal information transfer fromthe donor to the recipient ura3. Together, these results indicatethat the induced Ura⁺ His⁺ colonies that arose in the absenceof Rad51 were bona fide gene conversion products.

Rad51-independent DSB repair by allelic gene conversion is independent of Rad55, Rad57, and Dmc1. Yeasts encode two mitotic proteins related to Rad51, the paralogsRad55 and Rad57. Like Rad51, Rad55 and Rad57 bind DNA and areATPases, raising the possibility that the paralogs are responsiblefor the residual gene conversion in the absence of Rad51. BecauseRad55 and Rad57 function as a heterodimer (53), we first testeda rad51 ${Delta}$ rad55 ${Delta}$ double mutant. As shown in Fig. 3A, GALHO inductionled to similar levels of Ura⁺ His⁺ products in the rad51 ${Delta}$ mutantand the double mutant. To test whether Rad57 was responsiblefor the induced gene conversion in the rad51 ${Delta}$ rad55 ${Delta}$ double mutant,we tested a rad51 ${Delta}$ rad55 ${Delta}$ rad57 ${Delta}$ strain. As shown in Fig. 3A, DSBrepair by gene conversion in the triple mutant was similar tothat in the rad51 ${Delta}$ mutant and the rad51 ${Delta}$ rad55 ${Delta}$ double mutant.Dmc1 is more closely related to Rad51 than to Rad55/Rad57, butDmc1 is expressed only in meiosis (induced by nitrogen starvation).However, because allelic DSB repair by gene conversion occursat such low levels, it was possible that the rare HR eventswere occurring in a subpopulation of cells experiencing metabolicstress, resulting in transient Dmc1 expression and an HR-competentstate. We tested this with a rad51 ${Delta}$ dmc1 ${Delta}$ double mutant and againfound that the efficiency of DSB-induced gene conversion wassimilar to that in the rad51 ${Delta}$ mutant (Fig. 3A). Thus, Rad51-independent,DSB-induced allelic gene conversion is independent of Rad55,Rad57, and Dmc1.

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FIG. 3. Rad51-independent DSB repair by gene conversion requires Rad52 but is independent of Rad55, Rad57, and Dmc1. (A) Average Ura⁺ His⁺ frequencies (plus SD) are plotted for three or four determinations per strain incubated at 30°C. Significant differences between spontaneous (GALHO not induced) and DSB-induced frequencies are indicated by an asterisk (P < 0.03) or a double asterisk (P < 0.001). The rad52 ${Delta}$ strain is TP3828. (B) Data plotted as in panel A for cells incubated at 23°C; four determinations per strain. (C) Average Ura⁺ and Ura⁺ His⁺ frequencies in a second rad52 ${Delta}$ strain (RS3830) plotted as in panel A.

Rad51-independent allelic gene conversion is not inhibited at low temperature. DSB repair deficiencies caused by mutations in RAD55 or RAD57are more severe at 23°C than at 30°C. These cold-sensitivephenotypes are thought to reflect a greater requirement forRad55/Rad57 mediator function at lower temperatures becausethey are suppressed by overexpression of Rad51 and by mutationsin Rad51 that increase the affinity of Rad51 for DNA (7, 13,23). Because Rad55/Rad57 are thought to function only in Rad51-dependentDSB repair, we expected that Rad51-independent DSB repair bygene conversion would be insensitive to reduced temperature.We tested this by measuring DSB-induced Ura⁺ His⁺ frequenciesat 23°C in the rad51 ${Delta}$ mutant alone and in combination withthe rad55 ${Delta}$ and rad57 ${Delta}$ mutants. As shown in Fig. 3B, the lowertemperature had no significant effect on the level of Rad51-independentgene conversion in rad51 ${Delta}$ or rad51 ${Delta}$ rad55 ${Delta}$ strains, and while therewas a twofold reduction in the rad51 ${Delta}$ rad55 ${Delta}$ rad57 ${Delta}$ triple mutantcompared to the rad51 ${Delta}$ mutant, DSB-induced gene conversion inthe triple mutant was significantly higher than the spontaneouslevel. These results indicate that Rad55, and to a large extentRad57, functions mainly in the Rad51-dependent HR pathway.

Rad51-independent DSB repair by allelic gene conversion requires Rad52. Unlike Rad51, Rad52 has been shown to be essential for HR repairof DSBs in many substrates (17). We employed the selective ura3HR assay to determine whether we could detect a low level ofchromosomal DSB repair resulting in gene conversion in the rad52 ${Delta}$ mutant. Initial measurements indicated that GALHO inductionled to a slight increase ( $~$ 2-fold) in Ura⁺ His⁺ products, butthe difference was not statistically significant (Fig. 3A).There was wide variation in the frequencies of Ura⁺ His⁺ productsunder both growth conditions, with standard deviations approaching100% of the averages. This is consistent with most productsarising spontaneously, with variability reflecting differentialtiming of spontaneous events during population expansion ("jackpots").Fluctuation analysis is appropriate for measuring spontaneousHR rates, as it effectively eliminates problems associated withjackpots, but it is not suitable for measuring DSB-induced HR.In subsequent experiments with the original rad52 ${Delta}$ strain (TP3828)and with an independently constructed rad52 ${Delta}$ strain (RS3830),we continued to observe modest increases in Ura⁺ His⁺ frequenciesupon GALHO induction (data not shown). In cell populations witha low background of spontaneous products, GALHO induction ledto statistically significant increases in both total Ura⁺ andUra⁺ His⁺ products (Fig. 3C). Thus, there appears to be a lowlevel of Rad52-independent DSB repair by gene conversion, butit is sometimes masked by spontaneous jackpots. This resultis consistent with our previous report showing a small (statisticallysignificant) increase in DSB-induced gene conversion betweenura3 direct repeats in a rad52-1 mutant (P < 0.02 in bothMAT ${Delta}$ and MAT ${alpha}$ strains) (34). Both spontaneous and DSB-inducedevents are dependent on Rad51, as none were observed in therad51 ${Delta}$ rad52 ${Delta}$ double mutant (<6 x 10^–9) at 30°C (Fig.3A) or 23°C (data not shown). Together, the results indicatethat Rad51 is required for both spontaneous and DSB-inducedgene conversion in the absence of Rad52, and vice versa.

In the absence of Rad52, DSBs at MAT in haploid cells are usuallylethal, with rare survivors resulting from imprecise NHEJ (29).In diploids, DSBs at MAT lead primarily to loss of the brokenchromosome, but rare repair products arise via nonreciprocalcrossing over (half-crossover) with loss of the second chromosome(25). In contrast, spontaneous gene conversion with retentionof both chromosomes has been detected in rad52 mutants (8).We next investigated how Ura⁺ His⁺ and Ura⁺ His^– productsarise in the absence Rad52, with or without GALHO induction.With this ura3 allele arrangement, Ura⁺ His⁺ products cannotarise by half-crossovers without associated gene conversion(Fig. 4A), and Ura⁺ His⁺ products induced by DSBs cannot ariseby BIR (Fig. 1), suggesting that this class forms by a differentmechanism. To determine whether Ura⁺ products arose by conservativegene conversion, we analyzed HIS3:telV, HO, X764, and B3' markersin 18 rad52 ${Delta}$ products and found a variety of product types (Fig.4B). The majority of products from both uninduced and GALHO-inducedcultures were heterozygous at one or more ura3 markers and/orHIS3 (7 of 8 and 8 of 10, respectively), ruling out the half-crossover/chromosomeloss mechanism described by Malkova et al. (25). Note that BIRand half-crossovers differ in that the latter yields only onefull-length chromosome. Unlike wild-type cells, in which 100%of GALHO-induced products convert the HO site (35), there wasa modest (2:1) bias in favor of conversion of the HO allelein GALHO-induced rad52 ${Delta}$ cultures, consistent with the relativelylow level of DSB-induced gene conversion in the rad52 ${Delta}$ mutant.Although a similar twofold bias in HO site conversion was observedin the rad52 ${Delta}$ diploid when GALHO was not induced (Fig. 4B), thismay reflect leaky GALHO expression or sampling error. In bothrad51 ${Delta}$ and rad52 ${Delta}$ strains, DSBs are usually not repaired and chromosomeloss (yielding Ura^– His^– products) is the most commonoutcome.

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FIG. 4. Gene conversion in the absence of Rad52. (A) Half-crossovers can occur in any of the three intervals and in either direction, producing six potential products, none of which are Ura⁺ His⁺. (B) Marker patterns in Ura⁺ products from rad52 ${Delta}$ . The parent strain is heterozygous at the four indicated markers (gray boxes, signifying HIS3:telV, HO432, X764, and B3') and products from glucose (Glu) and galactose (Gal) cultures (uninduced/GALHO induced) show LOH at one or more markers. Conversions of the HO or X764 chromosomes are indicated by white and black boxes, respectively. Product types 4 and 7 showed no heterozygosity and may therefore reflect chromosome loss subsequent to conversion of HO or X764.

Crossovers occur in the rad51 ${Delta}$ mutant, but most large-scale LOH occurs by BIR or half-crossovers with the same genetic dependencies as Rad51-independent gene conversion. In wild-type cells, the majority of allelic DSB repair occursby gene conversion without an associated crossover. In the ura3heteroallele system employed in this study, conversion withoutcrossing over produces Ura⁺ or Ura^– recombinants thatretain heterozygosity at the HIS3:telV marker (His^+/–;phenotypically His⁺). Crossovers can be detected as either His^–products or, among His⁺ products as those that gain a secondcopy of HIS3:telV (His⁺⁺). Only His⁺⁺ products can be unambiguouslyattributed to crossovers because BIR or half-crossovers canalso produce His^– products. Crossovers are relativelyrare among mitotic gene conversions in wild-type cells (25,35), and only $~$ 5% of induced Ura⁺ colonies are His^– (Fig.5A). In the rad51 ${Delta}$ mutant, this percentage increased by sevenfold,and His^– fractions were similarly increased in doubleand triple mutants with rad51 ${Delta}$ , rad55 ${Delta}$ , rad57 ${Delta}$ , and dmc1 ${Delta}$ (Fig.5A). To distinguish whether the His^– products arose byfull crossovers or BIR/half-crossovers, we analyzed DSB-inducedUra⁺ His⁺ products from the rad51 ${Delta}$ and rad51 ${Delta}$ rad55 ${Delta}$ rad57 ${Delta}$ strainsand found that 1 of 17 and 1 of 21 products, respectively, wereHis⁺⁺ (data not shown). In the wild type, a similar fractionof Ura⁺ His⁺ products were His⁺⁺ (2 of 20) (35). Thus, crossoversare associated with gene conversions at similar frequenciesin the wild type and the rad51 ${Delta}$ mutant. In addition, becauseonly $~$ 5% of induced Ura⁺ His⁺ products are His⁺⁺ crossovers inthe rad51 ${Delta}$ background, $~$ 95% of induced Ura⁺ His^– productsresult from BIR or half-crossovers. As with gene conversion,Ura⁺ His^– products arose at similar frequencies in therad51 ${Delta}$ single mutant and in double/triple mutant combinationswith rad55 ${Delta}$ , rad57 ${Delta}$ , and dmc1 ${Delta}$ (Fig. 5B). Although the Ura⁺ His^–level was a few times lower in the rad51 ${Delta}$ mutant than the rad51 ${Delta}$ rad55 ${Delta}$ double mutant (Fig. 5B), in a separate experiment in whichwe tested these strains in parallel, the levels were not significantlydifferent (P = 0.23) and DSB-induced Ura⁺ His^– productsarose in the rad51 ${Delta}$ mutant at a level significantly higher thanthe spontaneous level (P < 0.0001) (data not shown).

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FIG. 5. Rad51-independent BIR and gene conversion have the same genetic dependencies. (A) The percentage of Ura⁺ products that are His^– (BIR plus crossovers) arising after GALHO induction is increased in rad51 ${Delta}$ and rad52 ${Delta}$ mutants. In the wild type (WT), BIR is not detected and His^– products result from crossovers (35). (B) DSBs induce BIR/half-crossovers in rad51 ${Delta}$ single, double, and triple mutants, but only slightly in the rad52 ${Delta}$ mutant. The values in both panels are averages plus SD for three or four determinations per strain. *, P < 0.03, and **, P < 0.005 for wild-type versus mutant in panel A and for spontaneous (GALHO not induced) versus DSB induced in panel B.

The majority of Ura⁺ products in the rad52 ${Delta}$ mutant were His^–(Fig. 5A) and could have arisen by BIR, half-crossovers, orchromosome loss after conversion, but similar to Rad52-independentconversions, these events were very rare (Fig. 5B). The datain Fig. 5B were generated from cultures incubated at 30°C,and similar results were obtained at 23°C (data not shown).The extremely low level of chromosomal BIR in the absence ofRad52 is consistent with results obtained in plasmid-directedBIR assays (6, 18). No Ura⁺ His^– products were detectedin the rad51 ${Delta}$ rad52 ${Delta}$ double mutant. We conclude that Rad51-independentBIR/half-crossover shows the same genetic dependencies as Rad51-independentDSB repair by gene conversion (Fig. 3A and 5B) and that bothconservative (conversion) and nonconservative DSB repair mechanismsare more strongly reduced in the absence of Rad52 than in theabsence of Rad51.

Frequent discontinuous conversion tracts in the absence of Rad51. To further investigate the mechanism of Rad51-independent DSBrepair by allelic gene conversion, we analyzed donor and recipientalleles in 17 DSB-induced Ura⁺ His⁺ products from the rad51 ${Delta}$ mutant and 21 products from the rad51 ${Delta}$ rad55 ${Delta}$ rad57 ${Delta}$ triple mutant;not surprisingly, these spectra were quite similar, and thetwo sets were included in a "combined rad51 ${Delta}$ " spectrum for severalof the subsequent analyses. Conversion tracts of rad51 ${Delta}$ productswere compared to tracts in 45 wild-type Ura⁺ products (Fig.6). As in the wild type, donor loci remained largely unchanged:donor conversions occurred in 3 of 17 rad51 ${Delta}$ products, 0 of 21triple-mutant products, and 1 of 45 wild-type products (therewere no significant differences between the wild-type and rad51 ${Delta}$ ,the triple mutant, or the combined rad51 ${Delta}$ sets [all P > 0.05;Fisher exact tests]). The average tract length (± standarddeviation [SD]) of the combined rad51 ${Delta}$ spectrum was 1,320 ±1,237 bp; this is somewhat longer than the 895 ± 916bp in the wild type, but the difference is not statisticallysignificant (P = 0.076). Although there was a trend toward longertracts in the rad51 ${Delta}$ mutant, a significantly higher fractionof the combined rad51 ${Delta}$ products (8 of 38) had very short tracts(<200 bp), whereas none of the 45 wild-type tracts were <227bp (P = 0.012; Fisher exact test). Four rad51 ${Delta}$ products, butno wild-type products, converted just the HO site (tract length,<53 bp), which is also a significant difference (P = 0.04;Fisher exact test) (Fig. 6 and Table 1). Thus, the rad51 ${Delta}$ tractswere more widely distributed, with both very short and verylong tracts, whereas wild-type tracts were more tightly clusteredaround the mean (Fig. 7A). Given that rad51 ${Delta}$ products includedboth shorter and longer tracts, it is not surprising that frequenciesof marker conversions as a function of distance from the DSBwere similar for wild-type and rad51 ${Delta}$ products (Fig. 7B). Thetwo tract spectra showed similar frequencies of unidirectionaland bidirectional tracts, and both lacked 3' unidirectionaltracts (Table 1).

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FIG. 6. Conversion tracts of DSB-induced Ura⁺ products from the wild type and combined rad51 ${Delta}$ mutants. The map at the top shows marker positions in the donor (X764) and recipient (HO432) alleles above and below the line, respectively. All markers are phenotypically silent except HO432 and X764. Minimum lengths of conversion tracts are represented by black bars, and the number and percentage of tracts recovered are listed. Only recipient alleles are shown; donor alleles usually remain unchanged. All products converted the HO site, but X764 never coconverts in Ura⁺ products. Discontinuous tracts (D1 to D11) are shown below, including three rad51 ${Delta}$ products in which recipient and donor conversion tracts were discontinuous (D9 to D11); in these cases, both chromosomes are diagrammed, and donor conversion tracts are shown as gray bars. The single donor conversion among the 45 wild-type products was continuous with the recipient conversion tract (not shown). Wild-type data are from Nickoloff et al. (35).

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TABLE 1. Ura⁺ gene conversion tract distributions in wild-type and the rad51 ${Delta}$ mutant

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FIG. 7. Properties of conversion tracts in wild-type (WT) and rad51 ${Delta}$ strains. All data are derived from tract spectra in Fig. 6. (A) Tract lengths are more widely distributed in the rad51 ${Delta}$ mutant than in the wild type; tract lengths are minimum estimates. (B) Discontinuous tracts are more frequent in rad51 ${Delta}$ . *, P < 0.02 (Fisher exact test). (C) Percent conversion of each marker as a function of distance from the DSB. *, P = 0.04; **, P = 0.0012 (Fisher exact test).

A striking feature of the Rad51-independent products was thehigh frequency of discontinuous conversion tracts. In wild-typecells, only 4.4% were discontinuous (2 of 45), but 20 to 30%were discontinuous in the rad51 ${Delta}$ , triple-mutant, and combinedrad51 ${Delta}$ spectra (Fig. 7C). In most cases, tracts had a singlediscontinuity, but two of nine rad51 ${Delta}$ discontinuous tracts hadtwo discontinuities (types D10 and D11) (Fig. 6). Most tractdiscontinuities were present in the recipient allele, with thedonor allele retaining its parental markers. In three rad51 ${Delta}$ products, tracts were discontinuous because markers convertedin separate regions in donor and recipient chromosomes (typesD9 to D11) (Fig. 6). However, donor conversion did not alwaysresult in a tract discontinuity. For example, in the rad51 ${Delta}$ productD11 (Fig. 6), the conversion of the donor Stu667 marker wascontinuous with the recipient tract. Conversions of donor andrecipient markers reflect mismatch repair in hDNA, and whileconversion tracts may not reflect the full extent of hDNA (dueto restoration-type mismatch repair), tracts do represent theminimum extent of hDNA. Tract discontinuities can result fromthree sources: discontinuous mismatch repair in a continuousregion of hDNA, repair in multiple hDNA regions arising duringa single HR event, and independent HR events. The preponderanceof discontinuous tracts in the rad51 ${Delta}$ spectra, however, cannotbe explained by independent HR events (see below).

DISCUSSION

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DISCUSSION

Spontaneous HR in the absence of Rad51 or Rad52. Low-level spontaneous HR occurs in both rad51 ${Delta}$ and rad52 ${Delta}$ mutants.Because spontaneous HR is eliminated in the rad51 ${Delta}$ rad52 ${Delta}$ doublemutant, spontaneous Rad52-independent HR depends on Rad51, andvice versa. Spontaneous HR has been proposed to initiate atspontaneous DSBs, such as when blocked replication forks collapseat single-strand damage. However, Lettier et al. (18) characterizeda class of rad52 mutants that cannot repair DSBs by gene conversion,SSA, or BIR but show wild-type or higher spontaneous HR rates.These rad52 mutants also show wild-type levels of HR stimulatedby single-strand damage induced by UV. Because Rad52 foci areseen most often during S phase (18, 20) and spontaneous HR ismarkedly reduced in rad52 ${Delta}$ (17) (compare Fig. 2B and 3A), itappears that Rad52 plays a critical role in HR-mediated restartof blocked or stalled replication forks. A role for Rad52 inreplication-associated HR is further indicated by the observationthat Holliday junctions form spontaneously in S phase in a Rad52-dependentmanner but independently of Rad51 and its paralogs (62).

There is strong sequence conservation between yeast Rad52 andhomologs in higher eukaryotes, yet mammalian RAD52 is not criticalfor DSB repair by HR, perhaps because its DSB repair functionhas been taken over by BRCA2 (43, 56, 61). However, recent evidenceindicates that human RAD52 is highly responsive to hydroxyurea-inducedreplication fork stalling (J. Wray, J. Liu, J. A. Nickoloff,and Z. Shen, submitted for publication), suggesting that itsrole in replication may be conserved through evolution. Prokaryotesalso express proteins with Rad52-like activities, includingEscherichia coli RecT (9, 36) and RecFOR (30) and other prokaryotic/viralRad52 homologs (11). It will be interesting to determine ifthese proteins also function in restarting blocked replicationforks.

Given the evidence that spontaneous HR initiates when replicationforks encounter single-strand lesions, such as nicks or gaps(18, 44), how might Rad51 and Rad52 promote these HR eventswhen both or only one of these proteins is present? Stalledor collapsed forks may be restarted by mechanisms that can resultin HR, including sister strand invasion or fork regression,which forms a "chicken foot" structure (45). In the absenceof Rad51, Rad52 could promote strand annealing between single-strandedregions (2), and in the absence of Rad52, Rad51 may load ontossDNA inefficiently but, once loaded, may efficiently catalyzestrand invasion (52), thus accounting for the low-level spontaneousHR in rad51 ${Delta}$ and rad52 ${Delta}$ single mutants, but not in the doublemutant. The extremely low level of DSB-induced HR in the rad52 ${Delta}$ mutant also depends on Rad51 and may similarly reflect the lowefficiency of Rad51 loading onto ssDNA in the absence of Rad52.

Conservative DSB repair by gene conversion in the absence of Rad51. Rad51 plays a central role in mediating strand invasion duringHR. Although there have been a number of reports of Rad51-independentectopic HR, in most cases, these events can be explained asresulting from SSA, BIR, or a combination of these processes(1, 2, 14, 50). Rad51 was reported to be essential for DSB-inducedallelic gene conversion at MAT (25, 50). Although DSB inductionat MAT is very efficient, MAT switching is not selectable andtherefore has low sensitivity for detecting rare HR events inmutant cells. With a selective ura3 assay, we found that Rad51-independent,DSB-induced allelic gene conversion was >30-fold higher thanthe spontaneous level. This increase was observable becauserad51 ${Delta}$ also markedly reduces spontaneous HR (Fig. 2B), highlightingthe importance of Rad51 for all HR events. We observed $~$ 30% cleavageof the HO site in ura3 after a 6-h GALHO induction (unpublishedresults), which probably reflects limited accessibility of HOnuclease to its site in the ura3 chromatin context. The moreefficiently cleaved MAT locus lacks nucleosomes at the HO recognitionsite (57). If ura3 were cleaved with 100% efficiency, detectable(Ura⁺) HR would increase $~$ 3-fold to $~$ 2 x10^–5 in the rad51 ${Delta}$ mutant; this is 100-fold above the spontaneous level. Becausea selective assay is required to detect these low-frequencyevents, coconversion of the HO site and X764 (Ura^–) isnot detected. In the wild type, long-tract Ura^– productscomprise $~$ 66% of gene conversions (35), and our results indicatethat long tracts may comprise an even greater fraction of Rad51-independentconversions. Thus, the absolute efficiency of Rad51-independentgene conversion may exceed 10^–4, but this is still severalorders of magnitude less efficient than the wild type.

Rad55 and Rad57 probably evolved from Rad51 via gene duplication.Our results indicate that they have lost most or all strandinvasion activity and function mainly within the Rad51 HR pathway,consistent with a prior genetic analysis (41). We also ruledout a contribution from Dmc1 in Rad51-independent HR. Thus,Dmc1 appears to function only in meiosis, and the Rad51-independentmitotic gene conversions described here are not mediated byleaky Dmc1 expression in mitotic cells. Together, these resultsindicate that DSB repair by gene conversion can occur independentlyof the two known yeast strand invasion proteins and their familymembers, albeit with low efficiency.

BIR is a nonconservative DSB repair process that involves single-endedstrand invasion and extension to the end of the chromosome.Two BIR pathways have been defined (16, 46), and the more efficientpathway requires the same strand invasion proteins as gene conversion(Rad51, Rad52, Rad55, Rad57, and Rad54) (6). In wild-type cells,this single-end pathway is far less efficient than gene conversion(25, 35), despite the fact that conversion requires coordinationof two broken ends with a donor duplex. DSB repair by the lessefficient Rad51-independent BIR pathway appears to have an efficiencysimilar to that of Rad51-independent gene conversion (Fig. 3Aand 5B). This indicates that in the absence of Rad51, single-endedand coordinated two-ended events occur with roughly equal probabilities.Rad51-independent gene conversion and BIR are both independentof Rad55, Rad57, and Dmc1 but dependent on Rad52 (Fig. 3A and5B) and probably Rad59 (2). The MRX complex regulates end resectionat DSBs (38) and is required for Rad51-independent BIR (16,46), so it is likely that MRX is also required for Rad51-independentgene conversion. Rad51-independent gene conversion and BIR aretherefore likely to reflect Rad52-/Rad59-mediated strand annealing(2), perhaps between resected ssDNA at the DSB and transientsingle-stranded donor sequences formed during replication, transcription,or natural DNA "breathing." If Rad52/Rad59 efficiently annealssDNA, the rate-limiting step may be the rare occurrence ofssDNA in duplex donor DNA. If these events depend on ssDNA formingduring replication or transcription, Rad51-independent DSB repairmay be S-phase dependent or more efficient in highly transcribedgenes.

A striking feature of Rad51-independent gene conversion is theprevalence of discontinuous conversion tracts. These tractsreflect the extent of hDNA formation and repair of mismatcheswithin hDNA (35, 39, 58). Tract discontinuities may form byseveral mechanisms, including discontinuous mismatch repairin a single stretch of hDNA, with repair involving oppositestrands as templates or with alternating patches of repairedand unrepaired mismatches. In the latter case, unrepaired mismatchessegregate in the next mitotic division and one daughter cellwill display a discontinuous tract. Mismatches may escape repairif hDNA is partially unpaired (3). It is unlikely that discontinuitiesseen in the absence of Rad51 reflect multiple tracts from independentHR events because each event is extremely rare ( $≤$ 10^–5)and independent events are expected at <10^–10. Theincrease in discontinuous tracts coupled with increased tractlength in the rad51 ${Delta}$ mutant is reminiscent of tracts in XRCC3-defectivemammalian cells (3). We proposed that XRCC3 does not activelysuppress long conversion tracts but rather that XRCC3 stabilizesshort hDNA intermediates and that in the absence of XRCC3, longhDNAs are selected because of their increased stability (3).The long discontinuous tracts in the absence of Rad51 may resultfrom similarly unstable hDNA. However, this does not accountfor the very short tracts that arose in the absence of Rad51.It is possible that the very short- and long-tract productsarise by distinct mechanisms. Interestingly, a similar bimodaltract length distribution was observed in Rad51C-defective mammaliancells (31).

Crossovers pose a significant risk of large-scale genetic change,including LOH of entire chromosome arms, deletions, inversions,and translocations (33), and crossovers are more frequentlyassociated with long gene conversion tracts (12, 22, 55). Althoughthe frequencies of crossovers associated with gene conversionswere similar in the wild type and the rad51 ${Delta}$ mutant, only short(and long discontinuous) Ura⁺ tracts could be analyzed in therad51 ${Delta}$ mutant. If it were practical to analyze long-tract Ura^–products, the total crossover rate might be higher in the rad51 ${Delta}$ mutant than in the wild type. It appears that Rad51 contributesto genome stability by enhancing the efficiency of DSB repairby conservative gene conversion that produces relatively short,continuous conversion tracts. This minimizes localized LOH,as well as large-scale LOH and chromosome rearrangements associatedwith crossovers.

ACKNOWLEDGMENTS

We thank Rosa Sterk, Jennifer Clikeman, Kimberly Spitz, andYi-Chen Lo for expert technical assistance and James Haber andJustin Wray for helpful comments.

This research was supported by grant CA118357 (awarded to J.A.N.and Mary Ann Osley) and by the University of New Mexico Initiativesto Maximize Student Diversity program, funded by NIH NIGMS grantGM060201 (directed by M. Werner-Washburne).

FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131. Phone: (505) 272-6960. Fax: (505) 272-6029. E-mail: JNickoloff{at}salud.unm.edu

Published ahead of print on 26 November 2007.

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

REFERENCES

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