Multiple Pathways Promote Short-Sequence Recombination in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, null allelesof several DNA repair and recombination genes confer defectsin recombination that grow more severe with decreasing sequencelength, indicating that they are required for short-sequencerecombination (SSR). RAD1 and RAD10, which encode the subunitsof the structure-specific endonuclease Rad1/10, are criticalfor SSR. MRE11, RAD50, and XRS2, which encode the subunits ofM/R/X, another complex with nuclease activity, are also cruciallyimportant. Genetic evidence suggests that Rad1/10 and M/R/Xact on the same class of substrates during SSR. MSH2 and MSH3,which encode subunits of Msh2/3, a complex active during mismatchrepair and recombination, are also important for SSR but playa more restricted role. Additional evidence suggests that SSRis distinct from nonhomologous end joining and is superimposedupon basal homologous recombination.

INTRODUCTION

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Introduction

Eukaryotic genomes are characterized by an abundance of short,repetitive sequences (11). In the yeasts Saccharomyces cerevisiaeand Schizosaccharomyces pombe, recombination between dispersed,short sequences, such as tRNA genes and ${delta}$ elements, can giverise to genome rearrangements (56, 69). In humans, recombinationbetween Alu elements create genome rearrangements that are involvedin several diseases (15). These observations suggest that carefulcontrol of recombination between short sequences may be essentialfor the maintenance of eukaryotic genome stability.

In S. cerevisiae, homologous recombination occurs less frequentlyper unit length for sequences of less than 300 bp (1, 26, 65)and becomes extremely inefficient for sequences of less than30 bp (36), suggesting that short-sequence recombination (SSR)is distinct from general recombination. Mutations in severalDNA repair genes have been shown to selectively limit recombinationbetween short sequences, demonstrating that SSR is under separategenetic control. Often, the effects of these mutations can becorrelated with changes in the processing of recombination intermediates.For instance, certain mutations in genes encoding subunits ofthe nucleotide excision repair (NER) and transcription factorIIH (TFIIH) complexes increase SSR and block the degradationof the ends of broken DNA molecules (5, 6, 32, 35). Similarly,loss of RAD27, which encodes a structure-specific nuclease importantin DNA replication and repair (18, 23, 27, 34, 51, 64), increasesSSR and decreases the cleavage of single strands adjacent toheteroduplex DNA (44). However, the precise relationships betweenthese DNA processing defects and the control of SSR remain unclear.

To better understand how SSR is restricted, we need to developan understanding of how it is propagated. We have begun thisprocess by identifying the factors that are required for SSRand determining how they are related to one another. We hypothesizedthat these factors are most likely to be previously identifiedcomponents of the double-strand break (DSB) repair machinerythat play as yet undescribed roles in recombination betweenshort sequences. Several recombination genes were selected foranalysis because their null alleles confer phenotypes that seemedlikely to provide insight into the basis of SSR. RAD52 was selectedbecause the rad52-null mutant of budding yeast is defectivefor most homologous recombination (47), which would be helpfulin determining the involvement of the basal recombination machineryin SSR. RAD1 was chosen because the rad1- and rad10-null mutantsare particularly defective for recombination between short sequences,suggesting that the NER and recombination endonuclease Rad1/10(7, 17, 30, 53, 60, 61, 67, 70, 71, 76) is an important componentof SSR (5). MSH2 and MSH3, which encode subunits of the mismatchrepair apparatus (52), were picked because the msh2- and msh3-nullmutants display recombination defects that are similar to thoseobserved in rad1 and rad10 mutants (58, 66), suggesting thatthey could also be required for SSR. MRE11, RAD50, and XRS2,which encode the subunits of M/R/X, a complex that plays a rolein many processes related to DNA recombination and repair (20),were chosen because their null alleles confer defective degradationof broken DNA molecules (25, 43, 73), suggesting that they couldbe involved in SSR. Finally, HDF1 (yKU70) and DNL4 were selectedbecause they are required for the repair of DSBs by nonhomologousend joining (NHEJ) (38, 74, 77), which has been shown to rejoinbroken DNA molecules by using very short (1- to 4-bp) homologoussequences (31, 59), and might, therefore, be part of the SSRapparatus.

The effects of null alleles of these genes on SSR, both singlyand in various combinations, were used to determine their relevanceto SSR and their relationships to each other. We measured SSRby assaying DNA fragment insertion into genomic target sequences.These assays revealed that SSR is composed of at least two pathwaysthat are superimposed upon general homologous recombinationcontrolled by the RAD52 gene. One pathway is controlled by theRAD1 and RAD10 genes, and the other is controlled by MRE11,RAD50, and XRS2. Our results indicate that these pathways competefor the same class of intermediates. The MSH2 and MSH3 genesare also important for SSR and act as members of both pathways,but their significance is more limited than that of the othergenes. DNL4 and HDF1, however, play very small roles in SSR,suggesting that the apparatuses for NHEJ and SSR are essentiallyseparate.

MATERIALS AND METHODS

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Materials and Methods

Yeast plasmids and strains. All of the plasmids used in this study were constructed, maintained,and amplified by standard techniques (57). Plasmids and DNAfragments were transformed into yeast by electroporation. Allof the yeast strains used in our work are isogenic with W303-1A(70) and are listed in Table 1. Construction and maintenanceof yeast strains were done by established protocols (12). Constructionof the rad1::LEU2 (54), rad10::LEU2 (5), rad50::hisG (3, 4),rad52::TRP1 (63), msh2::hisG (52), hdf1::TRP1 (38), mre11::hisG(10), mre11-6 (75), and mre11-H125N (40) mutant alleles wasdone as previously described. The msh3::hisG strain was constructedby transforming yeast with a 5.8-kb SpeI/MscI fragment of theplasmid pLAY410 where the 3.8-kb hisG::URA3::hisG universaldisruptor (2) replaces a 1.2-kb segment of the coding sequenceof MSH3. The xrs2::TRP1 and dnl4::LEU2 alleles were constructedby transforming yeast with PCR fragments created with primersthat had 45 bp of sequence upstream or downstream from the openreading frame to be replaced plus 15 to 26 bp of sequence flankingboth sides of the TRP1 and LEU2 sequences in pUC-TRP1 and pUC-LEU2(55), which were the templates for PCR. The sequences of thexrs2::TRP1 primers were 5'-CATTTGAAATCGGTATAGATACTAACAACGCCATCGTCATCACTAAACGCGTCAGCGGGTGTTGGCGGG-3'and 5'-CAGTTAATGCCTCCAGCAACCTGTAGTGCTTCTTTAAACCGATCCGCACACCGCATAGGCAAGTGCAC-3'. The dnl4::LEU2primerswere 5'-AAAATAAAAATCTAGAACTGAAGGAAATAGTAACGGATTATTTAGGTAGCGGGTGTTGGCGGGTGTCGGGGC-3'and 5'-TAATGTACATATGTAGGATAGTATTAAATAAACTTCAAAAAAGGCGAATGGCGCGACGCGCCC-3'.Transformants were selected on medium lacking tryptophan orleucine. Trp⁺ and Leu⁺ transformants were screened by Southernblotting to determine in which clones gene replacement had successfullyoccurred (G. M. Manthey and A. M. Bailis, unpublished data).The sam2::his3::ura3::KAN-MX allele that was used as a targetfor DNA fragment insertion in our assays was constructed bytransforming yeast with a 5-kb XhoI/NotI fragment from pLAY346containing the sam2::his3::ura3::KAN-MX construct. Transformantswere selected on medium containing 200 µg of G418 perml and screened by Southern blotting (Manthey and Bailis, unpublished).The sam2::his3::ura3::KAN-MX construct contained a 1.5-kb BglII/EcoRIKAN-MX marker replacing a 60-bp StuI/ApaI fragment of the codingsequence in a 1.2-kb BamHI/BamHI URA3 clone, which, in turn,replaces a 60-bp BglII/BglII fragment of the coding sequencein a 1.3-kb BamHI/XhoI HIS3 clone that has been inserted intothe SalI site in a 950-bp EcoRV/XhoI SAM2 fragment. The lengthof the HIS3 sequence incorporated into the sam2::his3::ura3::KAN-MXtarget encompasses all of the HIS3 sequences on the four his3::URA3fragments used in our DNA fragment insertion experiments.

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TABLE 1. Yeast strains used in this study

DNA fragment insertion assay. The DNA fragment insertion assay used in this work is substantiallysimilar to those previously described (44). his3::URA3 DNA fragmentswere prepared by restriction endonuclease digestion of pLAY144(5), phenol extraction, and ethanol precipitation. PurifiedDNA fragments were used to transform yeast cells by electroporation,selecting for expression of the URA3 segment of the fragment.Efficiencies of transformation with the DNA fragments variedby twofold or less from strain to strain (data not shown). Insertionof the his3::URA3 fragments into alternative genomic targetswas distinguished by replica plating to appropriate dropoutmedia. The nature of the recombination events in selected, independentrecombinants was confirmed by DNA sequencing. Insertion intothe wild-type HIS3 allele at the HIS3 locus replaces 60 bp ofthe HIS3 coding sequence with the 1.2-kb URA3 marker and resultsin His^- G418^r Ura⁺ transformants. Insertion into the his3::ura3::KAN-MXallele at the SAM2 locus replaces a 1.5-kb KAN-MX marker with60 bp of the URA3 coding sequence and results in His⁺ G418^sUra⁺ transformants. Recombination at the URA3 locus replacesa point mutation in the ura3-1 allele by gene conversion andresults in His⁺ G418^r Ura⁺ transformants.

Stability of HO-digested plasmid. A more detailed description of the assay used to determine HO-digestedplasmid stability was published previously (5). Single coloniesof strains ABT151 (wild type), ABT380 (mre11::hisG), ABT381(mre11-6), and ABT382 (mre11-H125N) were used to inoculate 300ml of synthetic medium lacking uracil and tryptophan and containing3% glycerol and 3% lactate. Uracil was omitted to select forthe URA3 marker on pLAY97, a centromere-containing plasmid thatalso bears a 117-bp fragment of MATa containing the recognitionsite for HO endonuclease. Tryptophan was omitted to select forthe TRP1 marker on pGHOT (46), another centromere plasmid containingthe HO endonuclease gene linked to a galactose-inducible promoter.Glycerol and lactate were used as carbon sources to maintainthe expression of the HO gene in neither an induced nor a repressedstate. Cultures were grown at 30°C to a density of 5 x 10⁶cells/ml before a 50-ml aliquot was removed, and the cells werepelleted and frozen at -20°C. HO endonuclease expressionwas then induced by adding 25 ml of 20% galactose to the culture.After 30 min, the cells from another aliquot were collectedand frozen. The remaining culture was filtered through sterile0.2-µm nitrocellulose filters, and the cells were resuspendedin prewarmed synthetic medium lacking tryptophan and containing2% glucose. Uracil was provided because pLAY97 is frequentlylost after HO cleavage, and glucose was used to repress HO geneexpression. At regular intervals, aliquots were removed andthe cells were collected as described above. DNA prepared fromthe frozen cells was digested with the NcoI restriction endonucleaseand analyzed by Southern blot hybridization using ³²P-labeledpBluescript (Stratagene, La Jolla, Calif.), the backbone ofpLAY97. Hybridization patterns were visualized and quantitatedby phosphorimaging (Molecular Dynamics). The stability of HO-digestedpLAY97 was determined by comparing the levels of the 1.5- and3.5-kb NcoI- and HO-digested fragments with that of the NcoI-digested5.0-kb fragment. Stability of HO-digested DNA was plotted asthe log of the percentage of HO-digested DNA remaining versustime. Curves were generated by least-squares analysis. Half-livesof broken DNA molecules were reported for two separate determinations.

RESULTS

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Results

DNA fragment insertion assay for SSR. We previously used the insertion of DNA fragments of variouslengths into alternative genomic targets to describe the effectsof specific DNA repair mutations on SSR (44). The assay usedin this study was very similar to the previously used assaybut was adapted for use with haploid cells (Fig. 1). DNA fragmentsconsisting of a URA3 marker flanked by different lengths ofthe HIS3 sequence (his3::URA3, Fig. 1B) were transformed intowild-type or mutant cells. Fragment insertion into the HIS3locus on chromosome XV uses only the terminal HIS3 sequences,while insertion into the sam2::his3::ura3::KAN-MX allele atthe SAM2 locus on chromosome IV uses the entire his3::URA3 sequence(Fig. 1A). Total homology to the HIS3 locus varied over sevenfoldfrom the smallest to the largest his3::URA3 fragment, whilehomology to sam2::his3::ura3::KAN-MX varied by less than 65%(Fig. 1B). Therefore, changing the length of the HIS3 sequenceson the his3::URA3 fragments should have a significant effecton their insertion into HIS3 but much less of an effect on overallfragment length and insertion into sam2::his3::ura3::KAN-MX.This makes the ratio of fragment insertions into HIS3 and sam2::his3::ura3::KAN-MX(H^-/K^-) an appropriate gauge of the cell's ability to use theterminal HIS3 sequences for homologous recombination.

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FIG. 1. DNA fragment insertion assay. (A) Description of the assay. DNA fragments electroporated into haploid yeast cells are inserted by recombination into the HIS3 locus on chromosome XV or the his3::ura3::KAN-MX marker at the SAM2 locus on chromosome IV. The fragments align with HIS3 by using the terminal HIS3 sequences, whereas the entire sequence is used to align the fragment with his3::ura3::KAN-MX. Insertion at HIS3results in a His^- G418^r (Kan⁺) Ura⁺ cell. Insertion at sam2::his3::ura3::KAN-MX results in a His⁺ G418^s (Kan^-) Ura⁺ cell. (B) DNA fragment substrates. The DNA fragments used in the experiments are depicted. HIS3 sequences are represented by shaded boxes with sequence lengths marked above in base pairs. The URA3 sequence is 1,071 bp long. The four fragments total 1,198, 1,321, 1,519, and 1,974 bp, respectively.

DNA fragment insertion requires the basal DSB repair machinery. We investigated the effects of mutations in several DNA repairand recombination genes on his3::URA3 fragment insertion inan effort to define the factors required for SSR. Similar topreviously reported results (50, 60), we found that the centralrecombination gene RAD52 was required to observe the insertionof DNA fragments into either of the targets discussed above,as well as gene conversion of the ura3-1 allele at the URA3locus, as no Ura⁺ transformants were obtained with the rad52-nullmutant. Therefore, all recombination between the DNA fragmentsand the genome is RAD52 dependent. This suggests that, in ourassays, SSR requires general homologous recombination.

Genes encoding key subunits of several important DNA repair complexes are required for SSR. It was previously reported that his3::URA3 DNA fragments areinserted less efficiently into the HIS3 locus of rad1 and rad10mutant cells than into that of wild-type cells (60, 61). Thisestablished that RAD1 and RAD10 play a role in the insertionof DNA sequences into the genome by recombination. We have alsopreviously shown that decreasing HIS3 sequence length reducesfragment insertion into the HIS3 locus of rad1 and rad10 mutantcells more than that into wild-type cells (5). This indicatesthat the roles of RAD1 and RAD10 in recombination increase assequence length decreases. Similarly, in the present study,we found that the rad1-null mutation had a much greater impacton the H^-/K^- ratio obtained with the shortest his3::URA3 fragment(127 bp of HIS3 homology), a 29-fold reduction from the wildtype, than with the longest (903 bp of HIS3 homology), a 2.5-foldreduction (Fig. 2). Together, these results suggest that RAD1and RAD10 play a critical role in the maintenance of SSR.

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FIG. 2. DNA fragment insertion in wild-type and mutant cells. The his3::URA3 fragments were electroporated into wild-type cells (ABX425-6B) and rad1::LEU2 (ABX441-26A), msh2::hisG (ABX464-12A), and msh3::hisG (ABX545-12A) mutant cells. Ura⁺ transformants were screened for DNA fragment insertion into HIS3 or sam2::his3::ura3::KAN-MX by replica plating to medium lacking histidine or containing 200 µg of G418 per ml. The mean ratios of His^- G418^r (Kan⁺) Ura⁺-to-His⁺ G418^s (Kan^-) Ura⁺ transformants (H^-/K^-) were determined from a minimum of five independent trials with each fragment in each strain. Log₁₀ values of the mean ratios ± 2 standard errors were plotted. Each trial consisted of a minimum of 400 transformants.

Like RAD1 and RAD10, the mismatch repair genes MSH2 and MSH3facilitate the removal of nonhomologous sequences from the 3'ends of recombining molecules (66). However, this role is distinctfrom the role played by RAD1 and RAD10 because it is importantonly when the recombining sequences are 1 kb or less in length.In our analysis, the msh2- and msh3-null mutants displayed H^-/K^-ratios with all four his3::URA3 fragments that were not significantlydifferent from those obtained with the rad1 mutant (Fig. 2),indicating that MSH2 and MSH3, like RAD1 and RAD10, play criticalroles in propagating SSR.

MRE11, RAD50, and XRS2 code for the subunits of another complexthat has nuclease activity (19, 40, 72, 75) and plays multipleroles in the maintenance of genome stability (20). The H^-/K^-ratios obtained by inserting all four his3::URA3 fragments intothe genomes of the mre11- and rad50-null mutants (Fig. 3) werevery similar to the values obtained with the rad1-, msh2-, andmsh3-null mutants (Fig. 2), suggesting that MRE11 and RAD50are also required for SSR. Meanwhile, the ratio obtained byinserting the smallest fragment into xrs2-null mutant cellswas slightly (two- to threefold) higher (Fig. 3). This suggeststhat XRS2 may play an independent and less critical role inSSR than do MRE11 and RAD50 or that the M/R/X complex playsa more peripheral role in the maintenance of SSR than a complexthat includes only Mre11 and Rad50.

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FIG. 3. DNA fragment insertion into wild-type and mutant cells. The his3::URA3 fragments were electroporated into wild-type and mre11::hisG (ABX441-21A), rad50::hisG (ABX441-22D), xrs2::TRP1 (ABX441-64A), dnl4::LEU2 (ABX448-4D), and hdf1::TRP1 (ABX449-7C) mutant cells. Transformants were screened, and H^-/K^- ratios were determined and plotted as described in the legend to Fig. 2 and in Materials and Methods.

Several mre11 alleles have been isolated that encode proteinsthat lack one or more of the nuclease activities identifiedwith Mre11 in vitro (19, 40, 75). We chose two of these alleles,mre11-6, which encodes a protein missing DNA binding site A(75), and mre11-H125N, which changes an amino acid in one offour conserved phosphoesterase motifs (10, 40), to investigatethe potential role of Mre11 nuclease activity in SSR. Mre11-6was previously found to lack single-stranded DNA (ssDNA) endonuclease,3' $->$ 5' ssDNA exonuclease, and 3' $->$ 5' double-stranded DNA exonucleaseactivities (75), while Mre11-H125N, which was not assayed forthe latter two activities, did not display ssDNA endonucleaseactivity (40). Both the mre11-6 and mre11-H125N mutations blockmeiotic DSB processing and confer a mild methyl methanesulfonatesensitivity. In addition, the mre11-H125N mutation was foundnot to change mitotic DSB processing and rejoining, NHEJ, ortelomere maintenance (40). The mre11-null mutant is blockedfor both mitotic and meiotic DSB processing, exhibits profoundsensitivity to methyl methanesulfonate, and is defective forNHEJ and telomere maintenance (20). Therefore, both mutantsshare some characteristics with the mre11-null mutant but notothers, suggesting that Mre11 performs both nuclease-dependentand -independent functions.

We found that mre11-null and mre11-6 mutant cells presentednearly identical, reduced H^-/K^- ratios for all four DNA fragments,but the mre11-H125N mutant displayed a significant variation(Fig. 4). The mre11-H125N ratios observed with the three largestDNA fragments were very close to those of the other mre11 mutants;however, the ratio obtained with the smallest fragment was morethan 12-fold higher and was only 2-fold lower than the wild-typeratio. This indicates that the three mre11 alleles reduce recombinationwith the longer sequences equally but that mre11-H125N has muchless of an effect on recombination with the shortest sequence.This demonstrates that MRE11 plays genetically distinct rolesin recombination with the three longer sequences and the shortestsequence, which we operationally define as SSR.

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FIG. 4. DNA fragment insertion into wild-type and mutant cells. The his3::URA3 fragments were electroporated into wild-type and mre11::hisG mre11-H125N (ABX480-1A) and mre11-6 (ABX518-6C) mutant cells. Transformants were screened, and H^-/K^- ratios were determined and plotted as described in the legend to Fig. 2 and in Materials and Methods.

One possible explanation for the role of MRE11, RAD50, and XRS2in SSR could be that these recombination events resemble MRE11-,RAD50-, and XRS2-dependent NHEJ. Other laboratories have shownthat DNA fragments and plasmids can be inserted into the genomeby using little, if any, homology (31, 39, 59). DNA sequenceanalysis of recombination junctions revealed that most insertionevents involve 1 to 4 bp of homology between the ends of thefragment and the genome, similar to a predominant class of NHEJevents. In addition, these events are dramatically reduced inrad50 -null mutants (62), indicating that RAD50, and perhapsMRE11 and XRS2, is required for insertion of DNA molecules intothe genome by using very short lengths of homology.

In order to ascertain whether NHEJ is involved in SSR, we performedDNA fragment insertion assays with NHEJ-defective hdf1- anddnl4-null mutant strains. The H^-/K^- ratios obtained with themutants were similar to the wild-type ratio and nearly indistinguishablefrom each other (Fig. 3). The mutant ratios were slightly (two-to threefold) reduced with the smallest fragment, suggestingthat NHEJ makes, at most, a very small contribution to our SSRevents. Therefore, the roles played by MRE11, RAD50, and XRS2in SSR may not be related to their roles in NHEJ. Interestingly,the H^-/K^- ratios obtained with the largest fragment were slightly(twofold) elevated in the hdf1 and dnl4 strains, indicatingthat NHEJ subtly inhibits recombination between longer sequencesin our assay.

Complex interactions between important factors control SSR in budding yeast cells. A previous analysis indicated that rad1 and rad10 are epistaticwith respect to SSR, as are mre11, rad50, and xrs2 (Mantheyand Bailis, unpublished). This suggests that RAD1 and RAD10work together, as do MRE11, RAD50, and XRS2. We conducted anepistasis analysis to explore the relationship between thesetwo groups of genes and others with respect to SSR.

DNA fragment insertions into various single and double mutantswere compared. For example, the combination of the rad1-nullmutation with the mre11-null mutation led to lower H^-/K^- ratiosthan either single mutation (Fig. 5). The extent of the decreases,130-fold for the shortest fragment in the double mutant versus18- to 29-fold for the single mutants, indicates that combiningthe mutations has synergistic effects. Very similar resultswere obtained with the rad1 rad50 and rad1 xrs2 double mutants(data not shown).

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FIG. 5. DNA fragment insertion into wild-type and mutant cells. The his3::URA3 fragments were electroporated into wild-type; rad1::LEU2 mre11::hisG (ABX441-39A), rad1::LEU2 msh2::hisG (ABX478-20C), and mre11::hisG msh2::hisG (ABX479-9C) double-mutant; and rad1::LEU2 mre11::hisG msh2::hisG (ABX526-56D) and rad1::LEU2 hdf1::TRP1 (ABX449-11A) triple-mutant cells. Transformants were screened, and H^-/K^- ratios were determined and plotted as described in the legend to Fig. 2 and in Materials and Methods.

Other laboratories have previously shown that null alleles ofMSH2, MSH3, RAD1, and RAD10 are epistatic to one another withrespect to recombination, indicating that they are members ofthe same recombination pathway (29, 58, 66). Similarly, ourdata indicate that null alleles of MSH2 and RAD1 are epistaticto one another with respect to SSR because the H^-/K^- ratiosfrom the rad1 msh2 double mutant (Fig. 5) were not significantlydifferent from the single-mutant ratios (Fig. 2). Intriguingly,the msh2-null allele also had an epistatic relationship withthe mre11-null allele (Fig. 5). We further explored the relationshipsamong MSH2, RAD1, and MRE11 by assaying a rad1 mre11 msh2 triplemutant and found that the H^-/K^- ratios were not significantlydifferent from the rad1 mre11 double-mutant ratios (Fig. 5),indicating that the msh2-null allele has no effect on SSR whenRAD1, MRE11, or both are mutated.

Epistasis analysis revealed that the H^-/K^- ratios from the rad1hdf1 double mutant were slightly (twofold) lower for the twoshortest fragments than those from the rad1 single mutants (Fig.5). Since the fold effect of the hdf1 allele relative to thewild type is slightly greater (Fig. 3), the relationship betweenthe rad1 and hdf1 alleles with respect to SSR is most likelysynergistic, indicating that the function of RAD1 is in a separate,competitive pathway from HDF1. Unfortunately, the extreme genomeinstability of the rad50 hdf1 double mutant (unpublished results)prevented our analysis of the strain and determination of therelationship between NHEJ and MRE11-, RAD50-, and XRS2-dependentSSR.

Defective processing of broken DNA molecules correlates with SSR deficiency in mre11 mutant strains. Several laboratories have demonstrated that the mre11-, rad50-,and xrs2-null mutants all exhibit a reduced rate of exonucleolyticdegradation of broken DNA molecules (24, 25, 65, 73). In contrast,we previously showed that the rad1-null mutant displays no suchchange (5). Interestingly, we also found that combining therad1-null allele with the rad3-G595R allele, which slows degradation,leads to a synergistic decrease in SSR (5). We concluded thatblocking the degradation of the ends of DNA molecules reducedSSR in cells that lack Rad1/10 nuclease activity. This suggeststhat the decrease in SSR observed in the rad1 mre11 double mutantmay, in part, be due to a similar combination of defects.

We investigated the link between mitotic DSB stability and SSRin the three mre11 mutants described above by using an assayfor measuring the degradation of a single-copy plasmid followingdigestion in vivo with HO endonuclease (5). Similar to the resultspresented by others (73) the mre11-null mutant strain displayeda markedly decreased rate of decay of broken plasmid molecules(Fig. 6). The half-life of an HO-digested plasmid was nearlythreefold longer in mre11-null mutant cells (82 to 87 min) thanin wild-type cells (30 to 34 min). Its half-life in mre11-6mutant cells was similarly elevated (68 to 77 min). Similarto the results previously described by Moreau et al. (40), thestability of HO-digested DNA molecules in mre11-H125N mutantcells was closer to that of the wild type (37 to 44 min). Thedifferent half-lives in these strains could not be accountedfor by differences in the repair of the broken molecules byNHEJ (Manthey and Bailis, unpublished). Therefore, the mre11-nulland mre11-6 mutants that presented definitive SSR defects asdescribed above (Fig. 4) were also found to possess a defectin the exonucleolytic processing of broken DNA molecules, whilethe mre11-H125N mutant exhibited significantly less severe defects.

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FIG. 6. Stability of a single-copy plasmid following cutting by HO endonuclease in wild-type (WT) and mre11 mutant cells. (A) Autoradiogram of plasmid DNA linearized in vivo. DNA was prepared from the wild-type (ABT151) strain and the mre11::hisG (ABT380), mre11-H125N (ABT381), and mre11-6 (ABT382) mutant strains carrying plasmids pLAY97 and pGHOT before and at intervals after a brief (30-min) period of HO endonuclease expression. The DNA was digested with NcoI prior to agarose gel electrophoresis and blotting to a nylon membrane. pLAY97 bands were revealed by hybridization with ³²P-labeled pBluescript. Digestion of pLAY97 with NcoI before cutting with HO endonuclease yields a 5.0-kb signal. This becomes 3.5- and 1.5-kb signals after cutting with HO. Degradation of HO-cleaved pLAY97 was indicated by changes in the intensity of the 3.5- and 1.5-kb fragments relative to that of the 5.0-kb fragment. (B) Stability of HO-digested plasmid DNA. The stability of HO-digested pLAY97 in wild-type, mre11Æ, mre11-H125N, and mre11-6 cells was determined by dividing the sum of the 3.5- and 1.5-kb signals by the sum of the 5.0-, 3.5-, and 1.5-kb signals, as determined by phosphorimaging. The resulting values were plotted versus time.

DISCUSSION

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Discussion

This report is a partial description of the apparatus requiredfor SSR in budding yeast. In order to characterize the SSR machinery,we used a DNA fragment insertion assay to measure recombinationin response to changes in DNA sequence length in a variety ofDNA repair and recombination mutant strains. This assay waschosen because of the simplicity of changing DNA sequence lengthand the similarity of its mechanism to the rescue of brokenchromosomes by break-induced recombination (42, 45, 47). Similarto results from other laboratories (50, 60), we found that DNAfragment insertion is completely dependent on RAD52, which isrequired for most recombination in budding yeast cells (47).This suggests that the pathways that mediate recombination betweenshort sequences are superimposed upon the general recombinationapparatus.

Our previous work showed that rad1- and rad10-null mutants displayrecombination defects that worsen progressively with decreasingDNA sequence length (5). The results from our current experimentsconfirm this, demonstrating that RAD1 is required for SSR inbudding yeast (Fig. 2). Previously, we postulated that Rad1/10is required for cleavage of the chromosomal target strand displacedby the invading DNA fragment, which creates the opportunityto form a covalent joint between the DNA fragment and genomictarget by ligation (5). Symington and colleagues also suggestthat Rad1/10 may cleave D loops during plasmid gap repair (68).We further postulated that the requirement for Rad1/10-facilitatedcovalent joint formation is related to the length of the homologoussequences because the shorter the length of homology, the lessstable the heteroduplex connecting the fragment to the target.This instability might necessitate the formation of covalentjoints that tether the fragment to the genomic target beforethe heteroduplex can dissolve, aborting recombination.

The results of the epistasis analysis presented here (Fig. 5)also indicate that another multiprotein complex, M/R/X, whosesubunits are encoded by the MRE11, RAD50, and XRS2 genes, occupiesa pathway for SSR that acts upon the same class of intermediatesprocessed by Rad1/10 (22), although the possibility that theyact sequentially cannot be rigidly excluded (41). M/R/X playsa role in many cellular processes, including mitotic and meioticrecombination, telomere maintenance, NHEJ, and DNA damage signaling(9, 20), and has been shown to possess several nuclease activitiesin vitro (19, 40, 72, 75). However, Mre11 is the only subunitwith intrinsic nuclease activity and can be thought of as thecore of a modular nuclease whose activity can be modified byadding or subtracting additional subunits (48, 72). It is unclearwhich, if any, of the nuclease activities of M/R/X are requiredfor mitotic recombination in budding yeast. Nuclease-defectivemre11 alleles, including the mre11-H125N allele that conferredlittle effect on SSR in our studies (Fig. 4), were previouslyfound to have wild-type levels of mitotic recombination (40,68), suggesting that Mre11 nuclease activity is not generallyrequired. Similarly, genetic evidence suggests that Mre11 nucleaseactivity may not play a role in the exonucleolytic digestionof the 5' strands of DSBs, since several nuclease-defectivemre11 alleles, including mre11-H125N, minimally affect DSB processing(Fig. 6) (40). In contrast, every nuclease-defective mre11 mutantthat has been tested is also defective for DNA damage signaling(14), suggesting that some sort of nucleolytic digestion ofbroken DNA molecules may be required to elicit the DNA damagesignal in mitotic cells (33, 49). This indicates that the functionsof Mre11 in DNA damage signaling and SSR are genetically separate.

Regardless of whether M/R/X plays a direct or an indirect rolein the degradation of broken DNA molecules, we have observeda correlation between the effects of three different mre11 alleleson the exonucleolytic degradation of DNA ends and their effectson SSR. We found that both SSR and the stability of DNA endswere significantly altered in mre11-null and mre11-6 mutantcells, while neither was greatly altered in the mre11-H125Nmutant (Fig. 4 and 6). This might suggest that Mre11-mediateddegradation of DNA ends plays a role in the propagation of SSR.If this is true, however, it is unlikely to be the sole functionof Mre11 in SSR, as several TFIIH/NER mutants defective forthe degradation of DNA ends exhibit increased SSR (5, 6, 32,35). We favor an alternative scenario in which the decreaseddegradation in the mre11 mutants may be indicative of the failureof a whole suite of proteins to act at the DNA ends, includingthose that propagate SSR. In support of this notion, recentevidence suggests that RAD50 may be required to load Msh2 ontocertain recombination intermediates at the ends of DNA molecules(16).

Interestingly, the mre11-H125N mutant displayed a defect inrecombination with longer fragments that was very similar tothe defects observed in the mre11-null and mre11-6 mutants,while recombination with the shortest fragment was far moredefective in the mre11-null and mre11-6 mutants (Fig. 4). Theseresults suggest that MRE11 controls both general recombinationand SSR and that these controls are separate. Further, it suggeststhat the recombination phenotypes of the mre11-null and mre11-6mutants are due to defects in both controls.

The msh2 and msh3 single mutants had profound SSR defects, equivalentto those of the rad1, mre11, and rad50 mutants (Fig. 2 and 3),indicating that MSH2 and MSH3 play an important role in SSR.This suggests that the Msh2/3 complex may play a role in SSRthat is similar to the role proposed in other studies of recombination(58, 66). Consistent with these studies, we observed that themsh2 and rad1 mutations were epistatic to one another with respectto SSR (Fig. 5), suggesting that the Rad1/10 and Msh2/3 complexesoccupy the same pathway for SSR (22). Unexpectedly, we foundthat the msh2- and mre11-null alleles were also epistatic (Fig.5). Since the evidence suggests that Rad1/10 and M/R/X are inseparate pathways and loss of MSH2 had no impact on this relationshipin a rad1 mre11 msh2 triple mutant (Fig. 5), we speculate thatour SSR assays may involve two processes, a dominant processthat is dependent on Rad1/10, M/R/X, and Msh2/3 and a subsidiaryprocess dependent only on Rad1/10 and M/R/X. It will be of furtherinterest to discover whether MSH6 is involved in this control,as the Msh2/6 heterodimer has been shown to bind to recombinationintermediates (37). It will also be important to determine ifrecruitment of additional factors by either of the heterodimersis required for SSR (8).

We offer the following model for the control of SSR based onthe conclusions from current and previous experiments (Fig.7). We suggest that SSR can begin by either 3' or 5' strandinvasion of the genomic target sequences by the DNA fragment,generating two different types of substrates. We believe that5' strand invasion may occur because our previous studies ofSSR in rad27 mutant strains strongly suggest that undegraded5' ends frequently participate in heteroduplex formation (44).Further, our observations that several TFIIH/NER mutations thatretard 5' strand resection significantly increase SSR suggestthat 5' strand invasion may be important (32, 35). Followingstrand invasion, Rad1/10 acts to cleave the displaced genomictarget strand, as discussed previously (5). Recent in vitroexperiments by the Sung laboratory have shown that the Mre11-Rad50heterodimer can endonucleolytically cleave a stem-loop structureon the 5' side of the loop (72), which is the same polaritywith which Rad1/10 cleaves displaced DNA strands (7). We hypothesizethat M/R/X might cleave genomic target strands displaced bythe invading DNA fragment in a manner similar to that of Rad1/10,explaining the synergism between the rad1-null and mre11-nullmutations. Alternatively, M/R/X might facilitate the actionof another, as yet unidentified, nuclease. M/R/X has been shownto facilitate Dnl4-catalyzed end joining in vitro, suggestingthat it may act as a platform for various enzymatic activitiesat the ends of DNA molecules (13). Cleavage of the target strandpotentiates ligation of the fragment strand to the target eitherdirectly or subsequent to 5' strand resection. The formationof a covalent joint between the fragment and target should stabilizetheir relationship and may be the rate-limiting step in SSR.

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FIG. 7. Model for Rad1/10, M/R/X, and Msh2/3 action during SSR. The his3::URA3 fragments (black lines, HIS3 sequences; shaded lines, URA3 sequences) align with the HIS3 genomic target (solid black lines). Either the 3' or the 5' end can be used for Rad52-dependent strand invasion. (Step 1) Invasion by the 3' end displaces the target strand of like polarity and forms a short, unstable heteroduplex. (Step 2) Msh2/3 (C clamp) binds at the junction between homologous (HIS3) and nonhomologous (URA3) sequences and stabilizes the D loop. Rad1/10, M/R/X, or another, M/R/X-dependent, nuclease (scissors) cleaves on the stabilized 5' side of the D loop, creating a free 5' end that is susceptible to exonucleolytic degradation. (Step 3) Exonucleolytic degradation (pacman) brings the 5' end of the target strand in register with the 3' end of the invading 3' strand of the fragment. (Step 4) Ligation of the 3' end of the fragment creates a covalent link between the DNA fragment and the target that resists unraveling and termination of recombination. (Step 5) Strand invasion, nuclease cleavage, and ligation tether the fragment ends to the target sequences, completing fragment insertion. (Step 6) Invasion by the 5' end displaces the target strand of like polarity and forms a short, unstable heteroduplex. (Step 7) Msh2/3 (C clamp) binds at the junction between homologous and nonhomologous sequences. However, Rad1/10, M/R/X, or an M/R/X-dependent nuclease (scissors) cleaves on the opposite, 5', side of the D loop, where Msh2/3 may not have any effect. Endonucleolytic cleavage creates a ligatable end. (Step 8) Ligation of the 5' end of the fragment to the genomic target creates a covalent link that should prevent loss of the recombination event by heteroduplex unraveling. (Step 9) Subsequent nuclease cleavage and ligation or another round of strand invasion, nuclease cleavage, and ligation ties the remaining fragment ends to the target sequences, completing fragment insertion.

The epistasis results suggest that Msh2/3 plays a more limitedrole in SSR than do Rad1/10 and M/R/X but one that overlapsthose of both. We suggest that this may be due to an interactionbetween Msh2/3 and Rad1/10, M/R/X, or a M/R/X-associated nucleasefollowing 3' strand invasion but not 5' strand invasion (Fig.7). When the 3' strand invades, we have proposed that the nucleaseswould cleave the displaced target strand near the position homologousto the junction between the HIS3 and URA3 segments on the incomingfragment. We further suggest that Msh2/3 bound at the junctionbetween homologous and nonhomologous sequences, as suggestedby Alani and colleagues (16), may stabilize the proximal armof the D loop and facilitate endonucleolytic cleavage. Whenthe 5' strand invades, however, the nucleases would cleave thedistal arm of the D loop, opposite from where Msh2/3 is bound.At that position, Msh2/3 may not play a role in determiningthe efficiency of endonucleolytic cleavage.

The presence of an apparatus committed to the maintenance ofrecombination between short sequences but not long sequencessuggests that SSR may play an important role in S. cerevisiae.We suggest that SSR may be ideally suited for rescuing DSBscreated in the wake of replication fork failure by sister chromatidrecombination. Not requiring extensive heteroduplex formationmay increase the rate of these local interactions and minimizethe likelihood of potentially deleterious interactions withduplicate sequences elsewhere in the genome. Interestingly,a number of studies have linked the RAD1 and RAD50 genes tosister chromatid recombination, supporting a potential linkwith SSR (21, 28, 39). Future studies will focus on determiningwhich interactions among the components of the SSR apparatusare the most critical in budding yeast in order to develop abetter understanding of how eukaryotic cells maintain genomestability.

ACKNOWLEDGMENTS

This work was supported by U.S. Public Health Service grantGM57484 to A.M.B., as well as funds from the Beckman ResearchInstitute of the City of Hope and the City of Hope NationalMedical Center.

We thank J. McDonald, E. Alani, N. Kleckner, R. D. Kolodner,K. Shannon, D. T. Weaver, S. Moreau, L. Symington, D. Bressan,J. Petrini, J. Nickoloff, and B. Jensen for strains and plasmids.We also thank P. Sung, J. Petrini, J. Haber, and the membersof the Bailis laboratory for stimulating discussions. Finally,we thank D. Bishop and several anonymous reviewers for insightfulcomments on the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Division of Molecular Biology, Beckman Institute of the City of Hope, 1450 E. Duarte Rd., Duarte, CA 91010-0269. Phone: (626) 359-8111, ext. 64031. Fax: (626) 301-8271. E-mail: abailis{at}bricoh.edu.

REFERENCES

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