Minisatellite Variants Generated in Yeast Meiosis Involve DNA Removal During Gene Conversion

Alexander J. R. Bishop,* Edward J. Louis† and Rhona H. Borts† *Department of Cancer Cell Biology, Division of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, Massachusetts 02115 and †Genome Stability Group, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom Manuscript received November 13, 1998 Accepted for publication May 1, 2000

ABSTRACT Two yeast minisatellite alleles were cloned and inserted into a genetically deﬁned interval in Saccharomyces cerevisiae. Analysis of ﬂanking markers in combination with sequencing allowed the determination of the meiotic events that produced minisatellites with altered lengths. Tetrad analysis revealed that gene conversions, deletions, or complex combinations of both were involved in producing minisatellite variants. Similar changes were obtained following selection for nearby gene conversions or crossovers among random spores. The largest class of events involving the minisatellite was a 3:1 segregation of parental-size alleles, a class that would have been missed in all previous studies of minisatellites. Comparison of the sequences of the parental and novel alleles revealed that DNA must have been removed from the recipient array while a newly synthesized copy of donor array sequences was inserted. The length of inserted sequences did not appear to be constrained by the length of DNA that was removed. In cases where one or both sides of the insertion could be determined, the insertion endpoints were consistent with the suggestion that the event was mediated by alignment of homologous stretches of donor/recipient DNA.

ARIABLE number tandem repeats (VNTRs), such and the internal structure of the alleles recovered are V as minisatellites and microsatellites, have become different in the two cell types, indicating mechanistic valuable tools for genome mapping (Nakamura et al. differences in how they arise (Jeffreys et al. 1994; Jef- 1987), forensic identification (Gill et al. 1985; Jeffreys freys and Neumann 1997). Finally, the germline events et al. 1985a,c), and population analysis (Wetton et al. characterized do not appear to be clonally derived (Jef- 1987). VNTRs may vary both in length (number of re- freys et al. 1994). This observation is more consistent peat units) and in repeat unit sequences (Jeffreys et with a meiotic origin rather than an origin during the al. 1985b). Minisatellites are distinguished from micro- mitotic divisions of germline development. The exis- satellites by their larger repeat unit, ranging from 8 to tence of cis-acting elements that elevate rates only in 100 bp in length. The repeat units often exhibit se- germline tissue are also taken as evidence for a meiotic quence polymorphisms that distinguish one unit from origin of minisatellite size changes (Jeffreys et al. the others in the array (Wong et al. 1986, 1987; Jeffreys 1998a). Proposed mechanisms to explain changes in et al. 1990) such that different minisatellite alleles may minisatellite size include unequal crossing over (Smith consist of a different sequential order of repeat units. 1976; Jeffreys et al. 1998a,b), gene conversion (Jef- The combination of these variables can produce Ͼ90% freys et al. 1994), and replication slippage (Levinson observable heterozygosity (Wong et al. 1986, 1987). The and Gutman 1987a,b; Schlotterer and Tautz 1992). 36 base-pair repeat (36 BPR) of Saccharomyces cerevisiae Distinguishing between such mechanisms is often diffi- is a typical subtelomeric minisatellite with alleles that cult. Detecting an unequal crossover requires the recov- differ in the number, order, and sequence of repeat ery of both recombinant meiotic products. To detect units (Horowitz and Haber 1984). gene conversion, the nonreciprocal transfer of se- A number of lines of evidence suggest that the high quence information, such that alleles do not segregate level of allelic minisatellite polymorphism seen in hu- in a normal 2:2 pattern, requires the analysis of all four mans is meiotically derived (Jeffreys et al. 1994; Jef- meiotic products. In mammalian systems, segregants freys and Neumann 1997). Rates of change are much where the information of one allele is flanked by se- higher in germline cells than in somatic tissues such as quences from the other parental allele have been inter- blood (Jeffreys et al. 1994). The distribution of sizes preted to be the result of gene conversion. However, such recombinants may also arise from a double crossover or other more complicated events. Replication slippage during the premeiotic DNA synthesis may also Corresponding author: Rhona H. Borts, Department of Genetics, Uni- versity of Leicester, Leicester LE1 7RH, United Kingdom. cause changes in sequence arrangement, but without E-mail: [email protected] transfer of information between homologues. In most

Genetics 156: 7–20 (September 2000) 8 A. J. R. Bishop, E. J. Louis and R. H. Borts systems it is impossible to distinguish forward slippage ments by selecting for recombination events in the vicin- during replication from an event involving an intrachro- ity of the minisatellite inserts. Analysis of these events mosomal deletion by recombination (for an exception indicated that there is an association of changes in min- see Ray et al. 1988). isatellite size with crossing over and gene conversions. The present consensus from human and yeast studies Five genes were mutated to determine their effect on is that the mechanism by which minisatellites change minisatellite recombination events. Four encode com- size resembles gene conversion as characterized in the ponents of the mismatch repair system (Msh2p, Msh3p, fungi (Buard and Vergnaud 1994; Jeffreys et al. 1994, Mlh1p, and Pms1p), and the fifth encodes the RecQ 1998b; Appelgren et al. 1997, 1999; Debrauwere et al. homologue, Sgs1p (Watt et al. 1995, 1996; Lu et al. 1999). For example, studies of human minisatellites 1996). Mismatch repair proteins were chosen because have noted that alterations in minisatellites occur fre- they are involved in the repair of heteroduplex DNA quently at one end of an array, with few or no events during meiotic recombination (reviewed in Borts et toward the other end (Jeffreys et al. 1990, 1991; Arm- al. 2000; Crouse 1998) and in recombination between our et al. 1993). This clustering of events is reminiscent diverged sequences (Borts and Haber 1987; Borts et of the gradients of gene conversion (polarity) observed al. 1990; Chambers et al. 1996; Hunter et al. 1996; at many loci in fungi (reviewed in Paˆques and Haber Chen and Jinks-Robertson 1999). No changes in the 1999; Petes et al. 1991), where the frequency of events frequency of minisatellite alteration were found in any decreases with distance from the initiating double- of the mismatch repair mutants analyzed. This is in strand break (Nicolas et al. 1989; Sun et al. 1989; Cao et contrast to the significant destabilization found for a al. 1990). A putative hotspot that, when present, confers CEB1 minisatellite in a mismatch repair defective strain elevated levels of meiotic recombination has been iden- (Debrauwere et al. 1999). tified upstream of a human minisatellite and has been The effect of mutation in SGS1 was examined because proposed to be the site of initiation for minisatellite two of the three known human RecQ homologues, BLM changes (Jeffreys et al. 1998a). and WRN, are required for genomic stability (Rosin Due to the difficulties of characterizing recombina- and German 1985; Gebhart et al. 1988). Interestingly, tion events in humans, model systems where different there have been conflicting reports on the stability (Fou- human minisatellites have been inserted into yeast have cault et al. 1996) or instability (Groden and German been developed (Applegren et al. 1997, 1999; Deb- 1992) of minisatellites in cells from Bloom’s syndrome rauwere et al. 1999). These studies find that the types patients. No change in stability was observed in this of events recovered are similar to those found in humans mutant background. and have many of the attributes of meiotic recombination found using conventional alleles in yeast. For example, Appelgren et al. (1999) unequivocally demonstrate MATERIALS AND METHODS gene conversion and a polarity gradient, while Deb- Strains: All constructs were made in the following two deriva- rauwere et al. (1999) demonstrate that heteroduplex tives of the yeast strain Y55 (McCusker and Haber 1998): DNA is formed during minisatellite recombination. Y55.2172 ϭ⌬HO MATa lys2-d leu2⌬ ura3-n ade1-1 his6-1 can1 They further show that minisatellite recombination is and Y55.2203 ϭ⌬HO MAT␣ leu2⌬ ura3-n cyh2 met13-4. Genetic procedures: Yeast manipulations and media were dependent on the production of double-strand breaks Њ and that the frequency of double-strand breaks corre- as described (Rose et al. 1990). Strains were grown at 30 on YEPD or synthetic complete media lacking one or more lates well with the frequency of recombination. nutrient. Sporulation was performed at room temperature as We present here a model system for studying the gen- described (Hunter et al. 1996). eration of meiotic variants in a naturally occurring min- PCR amplification: All PCR amplifications followed the isatellite of the yeast, S. cerevisiae. Using a genetically same basic protocol. PCR amplification was performed in NH4 defined chromosomal interval containing the 36 BPR buffer (supplied by Bioline Ltd., London) with 3.5 mm MgCl2, 0.15 pMol of each primer, and 0.01 U/␮l of Taq polymerase minisatellite, we examined and mapped recombination (Bioline). The reaction was heated to 95Њ for 3 min, then events that occur both within and flanking these ele- cycled 30 times at 61Њ for 30 sec, 72Њ for 1.5 min, and 94Њ for ments. Sequence analysis was used to characterize re- 20 sec, with a final annealing of 2 min and extension at 72Њ for 2.5 min. The primer pair prA-F and prA-R have optimal arranged minisatellite arrays. By tetrad analysis we iden- Њ tified events resembling gene conversion, deletion (or annealing temperatures of 58.5 . For amplification with the primer pair prC-F and prC-R a longer extension time of 2 replication slippage), and a complex combination of min during the cycling phase was used. both. No reciprocal events were detected, supporting Plasmid constructs: Two plasmids, pRED40 and pRED55, previous findings that unequal crossing over is not the were created based on pMJ13 (Lichten et al. 1987). pMJ13 major mechanism of minisatellite change (Wolff et al. contains MATa, URA3, and LEU2 sequences. pRED40 and 1988, 1989; Jeffreys et al. 1990). Appelgren et al. (1999) pRED55 were derived from this plasmid by mutation of LEU2 at the Asp718 site (leu2-a) and of URA3 at the NcoI site(ura3-n) have recently reported similar results in their analysis as described (Borts and Haber 1989). of the MS32 minisatellite inserted adjacent to the LEU2 The pAB series of plasmids was derived from either pRED40 hotspot. We obtained additional minisatellite rearrange- or pRED55. Two 36 BPR alleles, 12 ϫ 36 BPR and 15 ϫ 36 Meiotic Minisatellite Variants 9

Figure 1.—The MAT-MAT nontandem duplication on S. cerevisiae chromosome III. pBR322 sequences are indicated by the thin line. The 36 BPR array (12mer, ᭤᭤᭤᭤; 15mer, ᭤᭤᭤᭤᭤) was inserted at the BamHI site (B). The distances (in base pairs) between the minisatellite insertion site and the nearest genetic markers are indicated. Restriction sites (HindIII, H; EcoRI, R; SalI, S; and XhoI, X) used in plasmid construction are indicated. The speciﬁc conﬁguration of mating-type, minisatellite, and auxotrophic markers for each diploid used is given in Table 1.

BPR, were amplified by PCR from the YЈ clones pEL-31 (Louis transformation (Borts et al. 1986; Geitz et al. 1992) into and Haber 1992) and p69-3Ј (similar to the previously de- Y55.2172 and Y55.2203. The general structure of the nontan- scribed HinfI 1-6 clone; Horowitz and Haber 1984), respec- dem duplicated MAT locus is illustrated in Figure 1 and the tively, using the primers prA-F (5Ј-GGC GGA AGA TCT relevant genotypes of specific diploids are listed in Table 1. TCT GCA GCA GTG ATG AGG ACA GC-3Ј) and prA-R (5Ј- Y55.2172 and Y55.2203 derivatives containing mutations in GCG GGA AGA TCT GAA TTC ATC TTT ATT GGC GTC MSH2, MSH3, MLH1, PMS1,orSGS1 were created using the CTC C-3Ј). These primers amplified sequences corresponding plasmids pII-2::7-7 (Reenan and Kolodner 1992), pGEM7- to 43 bp 5Ј prior to the 36 BPR array to 23 bp 3Ј after the Zf(ϩ)/⌬CX URA3(Hunter and Borts 1997), pmlh1⌬::URA3 array within the subtelomeric YЈ sequence. PCR products were (obtained from M. Liskay), pWBK4 (Kramer et al. 1989b; digested with BglII (underlined) and purified using Stratagene Hunter and Borts 1997), and pPW⌬sgs1 (Watt et al. 1995), (La Jolla, CA) PrimeErase Quick Columns. The 12mer deriva- respectively. These constructs utilize either URA3 or LEU2, tive was ligated into BamHI-digested pRED40 and pRED55 which are the markers used to select random spore events, to to create pAB3 and pAB7, respectively. Similarly, the 15mer disrupt the relevant gene. It was thus necessary to further derivative was used to produce pAB1 and pAB5. The orienta- disrupt the URA3 or LEU2 marker with LYS2. Plasmids con- tion of the 36 BPR array within each of the plasmid constructs taining LYS2 disruptions of URA3 or LEU2, pRED223, and was determined by digestion with either PstIorEcoRI, which pRHB142, respectively, were created and used for this pur- cleave prA-F or prA-R, respectively (double underlined). The pose. The mutant strains were then crossed with the minisatel- orientation of the 36 BPR was chosen such that after integra- lite constructs to obtain the appropriate segregants. tion into the yeast genome the arrays were oriented in the Tetrad analysis: Spore colonies from dissected tetrads were same direction as those in the native YЈ elements. The BamHI grown for 3–5 days at 30Њ. Tetrads containing four viable site of pBR322 has been shown previously to be very close to spores were examined. Genetic analysis was conducted by a site of double-strand breaks in constructs similar to the ones replica plating onto various synthetic complete media lacking used here (Wu and Lichten 1995). one nutrient. Mating phenotypes were determined by the use Yeast strain construction: MATa-URA3-LEU2-MATa and MAT␣- of tester strains of known mating type. URA3-LEU2-MAT␣ nontandem duplications were created by Random spore analysis: Random spores were prepared as

TABLE 1 Strains used in this study

Haploid Transforming Diploid parents plasmids Structure at MAT MATa URA3 leu2-a MATa dAB1 tAJB1/AJB4 pRED40/RED55 MAT␣ ura3-n LEU2 MAT␣ MATa ura3-n G-12mer LEU2 MATa dAB2 tAJB2/AJB5 pAB7/AB3 MAT␣ URA3 G-12mer leu2-a MAT␣ MATa ura3-n G-12mer LEU2 MATa dAB3 tAJB2/AJB6 pAB7/AB1 MAT␣ URA3 A-15mer leu2-a MAT␣ MATa ura3-n A-15mer LEU2 MATa dAB4 tAJB3/AJB5 pAB5/AB3 MAT␣ URA3 G-12mer leu2-a MAT␣ MATa ura3-n A-15mer LEU2 MATa dAB5 tAJB13/AJB6 pAB5/AB1 MAT␣ URA3 A-15mer leu2-a MAT␣ 10 A. J. R. Bishop, E. J. Louis and R. H. Borts described (Lichten et al. 1987) and grown for 3–6 days at DNA (Borts et al. 1986) and Southern analysis (Southern 30Њ on synthetic complete medium lacking arginine and con- 1975; Sambrook et al. 1989). taining cycloheximide (10 mg/liter) and canavanine (40 mg/ Screening for novel sizes: To determine the length of the liter). Uraϩ Leuϩ recombinant spores were selected on a simi- 36 BPR array, colony PCR amplification was performed (Por- lar medium that also lacked uracil and leucine. Individual ter et al. 1993) with the following modifications. PCR amplifi- clones were tested for mating phenotype and examined by cation was conducted on freshly grown yeast cells lifted from PCR to determine the size of the 36 BPR array. A nonmating a YEPD solid medium and resuspended in 5 ␮l of Millipore-Q phenotype is indicative of a reciprocal crossover, an unequal (Millipore, Bedford, MA) water. The cell suspension was heated crossover, coconversion of URA3 or LEU2 and a flanking MAT to 95Њ for 3 min, and then 15 ␮l of PCR reaction mix was added. locus, or meiosis I nondisjunction of chromosome III. Non- The primers used were prB-F (5Ј-GGC GAC CAC ACC CGT mating clones that contained nonparental-size 36 BPR arrays CC-3Ј;54bp5Ј to the array) and prB-R (5Ј-GAT GCG TCC GGC were also examined by Southern analysis (Southern 1975) GTA GAG G-3Ј;81bp3Ј to the array). A total of 6 ␮l of each to determine whether a reciprocal or unequal crossover event reaction was analyzed by agarose gel electrophoresis. resulted in the phenotype. Disomy for chromosome III was Sequencing: DNA was amplified using a biotinylated primer tested for by contour-clamped homogeneous electric field (indicated by an asterisk). Primer sets of prC*-F (5Ј-CGG GAT analysis (Hunter et al. 1996). ATC GTC CAT TCC G-3Ј) and prC-R (5Ј-AGG AAT GGT GCA Statistics: Recombination events in strains with and without TGC AAG GA-3Ј) or prC-F and prC*-R were used in 50-␮l .(␮g/␮l 0.1ف) the 36 BPR inserts (Table 1 and Figure 2) were compared reactions containing 0.5 ␮l of genomic DNA using a contingency G-test (Sokal and Rohlf 1969). The prC-F and prC-R anneal 225 bp 5Ј to the array and 237 bp frequencies of size changes in mutant strains were compared 3Ј to the array, respectively. The PCR product was isolated to that in wild type using a standard normal z-test. A goodness- using magnetic beads (Dynal, A.S., Oslo). The primer prB-F of-fit G-test was used to compare the expected frequency of or prB-R was annealed to the single-strand template containing changes in array size in selected Uraϩ Leuϩ recombinants to prC*-R or -F, respectively. Sequencing was then performed the observed frequency as described in the text. with Sequenase (Amersham Pharmacia Biotech, Little Chal- Southern analysis: All yeast constructs were confirmed by font, United Kingdom) as recommended, including modifica- an appropriate restriction endonuclease digestion of genomic tions suggested by Dynal, A.S.

Figure 2.—Recombination events in the MAT-MAT interval. The major classes of genetically detectable recombination events are illustrated. The symbols used are the same as in Figure 1. The rate of occurrence of each class is given for the indicated constructs. * For simplicity, only conversion to wild type for either UraϪ or LeuϪ is illustrated, although all possibilities were recovered. The class of “other” includes unequal sister chromatid exchanges, intrachromosomal deletions, conversions of MAT, and unequal interchromosomal crossovers. Meiotic Minisatellite Variants 11

RESULTS A3B2C3-A4B3 (underlined in Figure 3A). After 9A3, The 12mer and 15mer minisatellite arrays: The 36 the 12mer and the 15mer alleles display different repeat BPR (Horowitz and Haber 1984) of the YЈ subtelom- organization. A single nucleotide polymorphism (a G eric element (Horowitz and Haber 1984; Louis and in the 12mer and an A in the 15mer) 22 bp upstream Haber 1990, 1992) in S. cerevisiae is a polymorphic mini- of the repeat array allow the centromere proximal ends satellite. Two alleles of the 36 BPR array were cloned of the two alleles to be distinguished. and inserted into a genetically defined artificial interval Meiotic recombination within the construct: Analysis at the MAT locus on chromosome III of S. cerevisiae of Ura, Leu, and mating phenotypes identified four (borts et al. 1986) (Figure 1). The alleles were shown major classes of recombination events (Figure 2). These to consist of either 12 (12mer) or 15 (15mer) repeat include reciprocal crossing over between URA3 and units of the 36 BPR array by sequence analysis. Each 36 LEU2, reciprocal crossovers between either URA3 or BPR unit is composed of three 12-bp segment types, A, LEU2, and a MAT locus and gene conversion of URA3 B, and C, the order of which is invariant (Horowitz and or LEU2 with or without a crossover (Borts et al. 1984). Haber 1984) (Figure 3A). Sequence polymorphisms Insertion of the 36 BPR does not significantly alter the between each segment type allows a subclassification of distribution of these recombination events (Figure 2). the sequences (i.e., variants A1, A2, B1, B2, C1, C2, etc.; PCR analysis of the 36 BPR of all segregants from Table 2). The first 8 of the 36 BPR units (1A1B1C1– the heterozygous diploids identified 36 recombination 8A3B4C4) and the next A segment (9A3), 304 bp to- events involving the 36 BPR (Figure 2). The majority tal, are identical between the alleles (see Figure 3A). (86%) were 3:1 segregations of parental-size alleles. The Within this 304 bp is a tandem duplication, C1-A2B2B2- remaining five were changes in the number of repeats

Figure 3.—Structure of parental and rearranged arrays. (A) A schematic representation of the 12mer and 15mer 36 BPR array sequences by segment types (see Table 1). The regions of identity between the two parental alleles (1A1 to 9A3) are represented by a hatched line. The two 108-bp internal tandem repeats are underlined. A nucleotide polymorphism (G/A) 22 bp proximal to the array is indicated. (B) The sequences of the ﬁve rearranged arrays obtained from tetrad analysis. An arrangement of parental arrays that could have produced each novel array is suggested with the recipient DNA as the lower strand in each case. A suggested deletion (᭡) of recipient 12mer sequences is shown for NT5. It can be seen that the proposed alignments of the parental arrays overlap with each other in regions where they are homologous. These overlapping regions therefore represent regions of ambiguous origin. For NT4 and NT5 the overlapping regions of the parental arrays illustrated include base discrepancies (white X in black box) between a parental array and the recovered novel array. 12 A. J. R. Bishop, E. J. Louis and R. H. Borts

TABLE 2 Sequence variation in the repeat unit

Segment A B C Variant 1 CTAATGCCAGTA CCAATGCGACTA CCAACTCCAGCA Variant 2 CTAATGCTACTA CCACTGCCAGCA CCAACGTCAGGA Variant 3 CTAGTGCTACTA CCACTGAAAGTA TCAACGTCAGGA Variant 4 CTAGTGCGACTA CCACCGAAAGTA CCGACTCCAACA Variant 5 CTAATGCCACTA CCACTGCTAGCA CCAACTCCAACA Variant 6 CCACTGAAGGTA in the array. Seven of the tetrads with a simple 3:1 gene array size and sequences of the other. This indicates that conversion of the minisatellite were picked at random the conversion event terminated between the upstream for further analysis. Sequencing was used to identify the polymorphism and the allele-specific portion of the 36 nucleotide at the A/G polymorphism 22 bp upstream BPR array. The arrays associated with the nonparental of the array and in the last A-segment of the array that A/G polymorphism were sequenced and showed no distinguished the parental arrays. Three of the seven sequence alteration, which confirms that conversion en- (one associated with a crossover, one with a gene conver- compassed the entire allele. Examination of the se- sion of LEU2, and one with no other associated event) quences from other progeny in each tetrad gave no consisted of the parental configuration of A/G polymor- evidence for any reciprocal sequence changes. These phism and array. The four remaining events (one associ- results suggest that additional events, such as gene con- ated with a crossover, two with a gene conversion of versions leading to base changes that do not alter the LEU2, and one with no other associated event) had the size of the minisatellite insert, have gone undetected. A/G polymorphism of one parent associated with the Novel minisatellites recovered by tetrad dissection:

TABLE 3 Minisatellite rearrangements recovered

Crossover Overlap size Array name Diploid Sizea associationb (NT)c Positiond From tetrads NT1 dAB3 18mer No 119 4B3–7C1 NT2 dAB3 11mer No 71 6A3–8A2 NT3 dAB3 18mer No 119 4B3–7C1 NT4 dAB4 8mer No 20 4C1–5B3 NT5 dAB3 16mer No Complex From random spores NR1–5 dAB3 9mer Yes 119 1B1–4C5 NR6 dAB3 9mer Yes 119 1B1–4C5 NR7 dAB3 9mer No 119 1B1–4C5 NR8 dAB3 11mer Yes 36 8A3–9A3 NR9 dAB3 11mer Yes 18 5C2–6A4 NR10 dAB3 5mer No 20 4B3–5A5 NR11 dAB3 17mer Yes Complex NR12 dAB4 18mer Yes 119 4C1–7B3 NR13 dAB4 18mer No 119 4C1–7B3 NR14 dAB4 11mer Yes 71 8A2–6A3 NR15 dAB4 11mer Yes 52 9C4–11A2 NR16 dAB4 17mer Yes 108 4B3–7B3 NR17 dAB4 9mer Yes Complex NR18 dAB4 13mer Yes Complex NR19 dAB3 18mer Yes NDe NDe a Size of the novel array is given by number of 36 BPR units. b Crossover association was determined by the nonmating phenotype of the haploid. c Overlap of perfect homology between the aligned parental sequences. d Position of the overlap within the novel array sequences. e Not determined. Meiotic Minisatellite Variants 13

Figure 4.—A possible alignment of recipient 12mer and donor 15mer sequences that might produce the observed NT5 sequences are illustrated schematically. Base pair differences between the aligned parental sequences and novel array sequences have been noted (white 1 in black box represents one mismatch within a 12-bp segment). An arrangement of the 12mer sequence where 72 bp are looped out is illustrated. The site of the loop out may be up to 4 bp proximal or 6 bp distal from the position given in the diagram (detailed in the inset with the 10-bp direct repeat underlined). The resultant deletion in 12mer sequences gives no further incorrect nucleotides in comparison with NT5 until 8B2 position 5. The 15mer alignment still results in two incorrect bases in comparison with NT5, but these are obtainable by repair from the 12mer alignment.

The rate of occurrence of size changes of the 36 BPR 12A2 to 15B3) and arise by a transfer of information was 0.5% of meioses (5/1082 tetrads from heterozygous from a 15mer into a 12mer accompanied by the loss of diploids and 1/205 from homozygous diploids). In all one repeat unit. six cases the alteration was nonreciprocal. NT4 may have been the result of a simple deletion The sequences of the five novel arrays from the het- event mediated by 20 bp of homology either side of erozygous diploids are shown schematically in Figure 3 the deletion, a gene conversion, or replication slippage and are described below. In general, parental sequences during the premeiotic DNA replication. It is not possible can be aligned from unique sequences outside of the to determine how this novel array arose. Of note though, novel array until mismatches are reached. A summary it is possible to extend the parental sequence alignment of the parental configurations that can produce the beyond the first mismatch on one side by 65 bp until novel arrays is shown in Table 3. The origins of the subsequent mismatches are encountered (see Figure first few repeat units of each novel array could not be 3B). Assuming that such an extended homology existed, unambiguously determined because the first eight re- mismatch repair could remove the single mismatch. peat units and the subsequent A segment are identical Further alignment of the parental sequences in the between the two parental alleles. However, the origins other events did not reveal any extended sequence ho- of the distal sequences could be determined, which, mology. NT5 is a complex event and was probably de- together with the inclusion of the upstream sequence rived by a combination of insertion of 15mer sequences polymorphism, allow us to say that novel tetrads 1, 2, and into 12mer sequences accompanied by deletion and 5 (NT1, NT2, and NT5) are clearly interchromosomal mismatch correction. A detailed description of the event events while NT3 and -4 could have been derived from can be found in Figure 4. intrachromosomal or sister chromatid interactions. Novel minisatellites recovered by random spore anal- Novel tetrads 1, 2, and 3 are relatively simple events. ysis: To determine if there is an association between the The NT1 18mer was derived from a 15mer by gene generation of minisatellite rearrangements and other conversion and the insertion of three repeat units re- recombination events, 576 Uraϩ Leuϩ meiotic progeny sulting in a triplication of the 108-bp tandem repeat. were selected at random from each of the two heterozy- The upstream polymorphism exhibited a 3G:1A segre- gous diploids. Such recombinants are generated by a gation pattern indicative of a conversion event while crossover between URA3 and LEU2, a gene conversion the sequences from 12B3 to the end of the array repre- of either ura3-n or leu2-a (with or without an associated senting the unique part of the 15mer sequence segre- crossover), or an unequal crossover between the MAT gate 2:2. NT3 is similar to NT1 with the exception that loci (see Figure 2). The array size was determined for the conversion did not encompass the upstream poly- each spore analyzed. The mating phenotype was used morphism. NT3 could also arise as a slippage of 108 bp to distinguish simple gene conversions of either URA3 during replication rather than conversion. The 11mer or LEU2 from those associated with crossing over, as the NT2 can be derived by insertion of 15mer sequences latter would yield a haploid nonmating spore. Southern into a 12mer. Sequences from 8A2 to the end of the analysis was used to distinguish unequal crossovers be- array are unambiguously of the 15mer (cf. Figure 3A, tween the mating-type loci from reciprocal crossovers. 14 A. J. R. Bishop, E. J. Louis and R. H. Borts

Figure 5.—Eighteen novel array sequences from random spores are represented as in Figure 2. NR1–11 are derived from dAB3 and NR12–18 from dAB4. NR6 is exactly as NR1–5 with the exception that the associated upstream polymorphism is “G.” NR7 is exactly like NR1–5 with the exception that the associated upstream polymorphism is a G and that it has a MAT␣ phenotype. NR13 is as NR12 except that the associated upstream polymorphism is an “A” and that it has a MATa phenotype. As with Figure 3, possible alignments of the parental arrays that could give rise to the novel arrangements are indicated. A suggested deletion (᭢) is shown for NR16 and base discrepancies between the parental arrays and the novel array are indicated by (white X in black box). The majority of the recovered novel arrays have sequences at their ends consistent with a single event leading to the selected Leuϩ Uraϩ spore. In other words, they start with the 15mer upstream polymorphism and end with the 12mer array sequence for dAB3 and vice versa for dAB4. Within these are complex insertions of one array’s sequences into the other accompanied by deletion of sequences of the recipient array as discussed.

It should be noted that a gene conversion of URA3 or sequenced. This diploid contains MATa-(ura3-n)-(G- LEU2 that includes one of the flanking mating-type loci 12mer)-(LEU2)-MATa and MAT␣-(URA3)-(A-15mer)- could not be distinguished from a crossover between (leu2-a)-MAT␣ (Table 1). The simple expectation for URA3 and LEU2. However, the frequency of these long this configuration of markers is that a novel array would conversions has been shown to be very low (Borts and consist of an “A” characteristic of the 15mer parent and Haber 1987, 1989). the sequences from the distal portion of a 12mer. Eight From the 1152 Uraϩ Leuϩ haploid progeny exam- such recombinants were observed. The three remaining ined, 21 were identified that had a novel, nonparental- events had the upstream polymorphism and the unique size 36 BPR array. Two events involved an unequal cross- distal sequences of a 12mer (see NR6, 7, and 9, Figure over between MAT loci and were excluded from further 5 and Table 3). The seven novel arrays derived from analysis. Of the 19 (1.6%) remaining Uraϩ Leuϩ spores dAB4 were also sequenced. dAB4 consists of the arrange- containing novel size arrays all but three (NR7, NR10, ment of close flanking markers opposite to that of dAB3 NR13) were recombinant for the flanking mating-type (see Table 1). Of the seven events, four begin with loci. The sequences of the arrays are comparable to 12mer sequences and end with 15mer sequences (Fig- those observed in the tetrad analysis and are illustrated ure 5 and Table 3), as expected. Two, however, began schematically in Figure 5. Table 3 summarizes the char- and ended with 15mer sequences and one with 12mer acterization of the simple novel arrays. sequences. Of these three events, NR17 and NR18 (Fig- Eleven of the 12 novel size arrays from dAB3 were ure 5) were clearly interallelic as they contained allele- Meiotic Minisatellite Variants 15 specific sequences from both arrays. These recombi- difficulty of clearly establishing the nature of the recom- nants are therefore of a complex, patchy nature. In bination event in human systems, yeast has been ex- total, 14 of the 18 analyzed arrays are unambiguously ploited as a model organism to study these issues. A derived from an interchromosomal minisatellite inter- number of studies have recently been performed that action. take advantage of the ability to analyze all four meiotic Association between minisatellite rearrangements and products and that utilize the extensive range of recombi- recombination of flanking markers: To determine if nation mutants to better characterize the mechanism minisatellite size changes were associated with other by which minisatellites change size (Appelgren et al. recombination events, we measured their frequency in 1999; Debrauwere et al. 1999; this study). In one study, spores selected to be recombinant for the flanking URA3 size changes of the human MS32 minisatellite were iden- and LEU2 genes. If there were no association between tified among tetrads whose four meiotic products had an altered minisatellite and other recombination events, been pooled (Appelgren et al. 1999). When a change it would be expected that the frequency of size changes was identified, all four meiotic products were subse- would remain the same, independent of selection for quently analyzed. In another study, changes in size of other recombination events. From the tetrad analysis, the human minisatellite CEB1 were shown to be depen- we determined the frequency of size changes to be dent on the formation of a meiotic double-strand break, 0.12% (5 minisatellite changes in 4328 spores derived providing strong evidence for a recombinational mecha- from the heterozygous diploids). Among spores selected nism leading to changes in size (Debrauwere et al. to be Uraϩ Leuϩ recombinants the frequency was 1.6% 1999). A heteroduplex intermediate was also demon- (19/1152). This 13-fold enrichment of minisatellite strated and mismatch repair proteins were shown to changes demonstrates that there is a significant associa- affect the rate of size changes. tion between the changes in minisatellite array size and In the study reported here, we analyze all of the mei- recombination events that produce a Uraϩ Leuϩ pheno- otic products individually and fully sequence the size type (G ϭ 37.3; P Ͻ 0.001%). changes. This analysis indicates that minisatellites un- Novel minisatellites recovered by random spore anal- dergo a high rate of simple gene conversion, a type of ysis from mutant strains: Random spore analysis was event that would not have been detected in all previous carried out in five mutant strains. 576 Uraϩ Leuϩ spores studies of minisatellite recombination. We also detect from msh2 and 1152 from each of msh3, mlh1, pms1, the complex events found in other studies and demon- and sgs1 strains were analyzed (Table 4). No significant strate that they can be associated with crossing over. We difference in the rate of array size change was observed failed to detect an effect of mutation in any of the for any mutant background examined. Given the num- mismatch repair genes tested, in contrast to that seen ber of spores analyzed, a twofold increase in rate relative in the study of CEB1 (Debrauwere et al. 1999). to wild type would have been statistically significant. One observation unique to this study is that six times as many parental-size arrays segregated 3:1 than exhibited a change in size. Half of these 3:1 events were DISCUSSION associated with a crossover. Some events involved gene conversions that encompassed the entire array and up- Minisatellites can be highly polymorphic genetic ele- stream G/A polymorphism. Other events appear to en- ments. The mechanisms by which the observed allelic compass only one end of the array or the other. The differences are generated are beginning to be defined. preliminary examination presented here indicates that The present consensus is that minisatellites alter by a a simple conversion, as well as changes in size, may gene conversion mechanism (Jeffreys et al. 1994), which be associated with crossovers and/or extended gene may or may not be accompanied by recombination of conversion tracts. Jeffreys and colleagues have also re- flanking markers (Jeffreys et al. 1998a,b). Due to the cently reported exchange of flanking markers without a change in array size (Jeffreys et al. 1998a,b). Although TABLE 4 infrequent, such events may be similar to those reported here. Effect of mutation on the frequency of changes A second conclusion that can be drawn from the in size of the 36 BPR repeat tetrad analysis is that substantial amounts of DNA are lost from the recipient array concomitant with insertion Genotype Size changes (%) of the donor sequences. In the five events analyzed in Wild type 19/1152 (1.6) detail, the minimal amount of sequence lost (calculated msh2 14/576 (2.4) from the distance between the nearest sequences of msh3 24/1152 (2.1) unambiguous parental origin) ranged from Ն44 to mlh1 31/1152 (2.6) Ն149 bp. This loss of sequence is difficult to account pms1 14/1152 (1.2) sgs1 30/1152 (2.6) for by the repair of mismatched heteroduplex involving these sequences. 16 A. J. R. Bishop, E. J. Louis and R. H. Borts

An intriguing difference between the study reported Borts and Haber 1987; Rayssiguier et al. 1989; Borts here and that of the human minisatellites is that the et al. 1990, 2000; Chambers et al. 1996; Hunter et al. events identified in the 36 BPR were predominantly 1996). Such an effect was seen by Debrauwere et al. interallelic. Three out of five minisatellite rearrange- (1999) in pms1msh2 double-mutant strains containing ments detected in tetrads and 14 out of 18 of the random CEB1. They also recovered postmeiotic segregations of spore size changes clearly resulted from interallelic minisatellite sequences, the hallmark of unrepaired het- events. In contrast, studies of the two different human eroduplex. The increase in rate found in the pms1 msh2 minisatellites inserted into yeast show a bias toward in- CEB1 strain is consistent with either the recovery of traallelic events (Appelgren et al. 1997, 1999; Deb- previously undetected events resulting from sister chro- rauwere et al. 1999). Such a bias might be indicative of matid repair of rejected events or with the recovery of heteroduplex rejection or mismatch-repair-stimulated events destroyed by mismatch-repair-stimulated killing. secondary double-strand breaks of mismatched interho- In addition, data suggestive of a role for mismatch-repair- mologue events, both of might then preferentially use induced recombination in minisatellite size changes can intrachromosomal recombination to repair the break. be found in the type of events observed in the MS32 When a mechanism such as single-strand annealing is tetrads (Appelgren et al. 1999). Many of the events used to repair the break it can lead to a detectable size reported there appear to have arisen from multiple change. However, repair from the perfectly homologous rounds of recombination. Such events are consistent sister may not lead to a detectable change. The observa- with a model in which the repair of multiple mismatches tion that in the absence of mismatch repair Debrauw- induces a second recombinational interaction (Borts ere et al. (1999) recover twofold more events, half of and Haber 1987; Borts et al. 1990). which contain unrepaired heteroduplex, is consistent There are a number of possible explanations for why with this proposal. Debrauwere et al. (1999) found an effect of mismatch Another difference among the studies is the variation repair genes and none was found in this study. First, in rate of size changes detected, which ranged from Debrauwere et al. (1999) deleted both PMS1 and MSH2 0.12% (36 BPR) to 15.6% (MS32) of spores assayed. while the genes were deleted individually in this study. These differences may reflect the genomic location of It has been suggested that Pms1p and Msh2p may act the insertion (Goldman and Lichten 1996; Debrauw- at least partially independently to impair recombination ere et al. 1999) although other explanations are possi- in the presence of sequence heterozygosity (Chambers ble. For example, the 20- to 100-fold difference between et al. 1996) and thus it might be necessary to eliminate MS32 and the other minisatellites may reflect the fact both genes before an effect would be detectable. An- that the MS32 rate was measured in a RAD5 defective other possible explanation might be that secondary background (Fan et al. 1996; Appelgren et al. 1997). structures formed by the 36 BPR minisatellite during the RAD5 encodes a helicase involved in microsatellite insta- recombination process might be insensitive to mismatch bility (Johnson et al. 1992) and potentially involved in repair proteins. For example, there is a 15-bp imperfect the mechanism of minisatellite changes. inverted repeat contained within the 36 BPR unit We found no effect on the rate of minisatellite repeat (Horowitz and Haber 1984). Palindromic sequences size changes in any of the mismatch repair (pms1, msh2, have long been known to escape mismatch repair (Nag msh3, mlh1) or helicase (sgs1; Watt et al. 1995, 1996; et al. 1989; Nag and Kurst 1997) presumably because Lu et al. 1996) mutants studied. Interestingly, it has they form hairpin structures that are not recognized by recently been shown that the absence of Sgs1p stabilizes the mismatch repair system. In addition, single-strand an unstable microsatellite (fragile X) inserted into yeast loops of the size postulated to account for many of the (White et al. 1999) during vegetative growth. Although events (Figure 6F) are known to be partially (Kirk- our study is on meiotic minisatellite instability, the lack patrick and Petes 1997) or completely (Williamson of an effect of the sgs1 mutation on the 36 BPR supports et al. 1985; Kramer et al. 1989a) insensitive to the mis- the notion that the instability of microsatellites and min- match repair system. It is an intriguing possibility that isatellites may be controlled by different genes (Sia et mechanisms of size change may be sequence or struc- al. 1997; Kokoska et al. 1998). ture dependent and thus may vary from minisatellite to The absence of an effect of mismatch repair mutants minisatellite. on the rate of change of the 36 BPR was unexpected. The results presented above and those from the other The sequences present in the minisatellite alleles will minisatellites studied, contribute to our understanding generate highly mismatched heteroduplex DNA. Be- of the mechanisms by which minisatellites change size. cause highly mismatched DNA has been shown to be The clear interhomologue derivation we see for the subject to processes that reduce the frequency of inter- majority of size changes is incompatible with replication chromosomal recombination (heteroduplex rejection, slippage during the premeiotic DNA synthesis. This, mismatch-repair-stimulated killing) the prediction is combined with the observation that size changes are that recombination frequencies would be elevated in dependent on Spo11p-mediated double-strand breaks the absence of mismatch repair (Doutriaux et al. 1986; (Debrauwere et al. 1999), lead us to favor recombina- Meiotic Minisatellite Variants 17

Figure 6.—Generation of minisatellite rearrangements by double-strand break repair. After pre-meiotic DNA replication one of the four chromatids suf- fers a double-strand break and the ends are processed by a 5Ј to 3Ј exonuclease that generates two single-strand 3Ј DNA tails (A). One 3Ј single-strand end can invade homologous DNA (B) and serve as a primer for DNA synthesis. In the double-strand break repair model (Szostak et al. 1983), synthesis displaces one strand of the invaded DNA duplex, making it available to the other 3Ј single- strand end (C). Once the displaced strand “captures” the other 3Ј single- strand end by annealing, it could prime DNA synthesis (D) and thus generate a double Holliday junction (E). In the synthesis-dependent single-strand annealing model (Hastings 1988) following invasion (B) and synthesis, the newly synthesized DNA disassociates from the invaded chromatid and anneals with the other 3Ј single-strand end (CЈ). In both models insertion of nonparental sequences into the recipient can occur by misalignment of repeat units between the displaced single- strand DNA and the 3Ј single-strand end. This can result in unpaired “tails” that must be excised (arrowhead, D, DЈ) before synthesis can repair the break (E, EЈ) and leads to loss of sequence from the recipient. Simple single-strand annealing between repeats (BЈ) can occur following initiation and exposure of the 3Ј single-strand ends (A). This necessarily results in an intrachromosomal deletion (BЈ). Misaligned annealing can also generate looped structures such as illustrated in F. Cleavage (arrowhead) of the loop leads to deletion of sequences such as are recovered in NT5. tion as the mechanism by which minisatellites change conversions where parental-size minisatellite arrays seg- size. The absence of an effect of mutation in mismatch regated 3:1 with or without an associated crossover (Fig- repair genes leads us to propose a model in which the ure 6E). formation of heteroduplex DNA occurs only within se- One observation made in this study not readily ac- quences of perfect or nearly perfect homology. Figure counted for by the conventional models is the net loss 6 illustrates a version of the canonical double-strand of repeats in the recipient arrays (Table 3) detected in break model (Szostak et al. 1983) as well as a version of the tetrad analysis. This loss of sequence is difficult to synthesis-dependent single-strand annealing (Hastings explain by simple heteroduplex formation and repair 1988; reviewed in Paˆques and Haber 1999). We pro- of single base mismatches. However, the loss of recipient pose, as do Debrauwere et al. (1999), that both mecha- DNA could occur at a number of points in the recombi- nisms contribute to changes in size of minisatellites. In nation process. For example, sequences could be re- both models recombination is initiated by a double- moved by double-strand exonuclease activity at the time strand break in what will be the recipient DNA molecule. of the breakage by a 3Ј exonuclease activity acting on This is followed by the removal of one strand by a 5Ј to the noninvading strand. Another possibility is the degra- 3Ј exonuclease to generate 3Ј single-strand tails (Sun et dation of the invading 3Ј end either prior to or in al. 1991; reviewed in Paˆques amd Haber 1999). In a conjunction with heteroduplex formation. These latter repeated structure, an intrachromosomal deletion can mechanisms all result in gap formation (Szostak et al. occur by single-strand annealing as is often observed in 1983). Although there has been no physical evidence mitosis (Galli and Schiestl 1995) (Figure 6, BЈ). This that either 3Ј tail is degraded using homologous sub- type of event economically explains the intrachromo- strates (Liu et al. 1995), no experiments have ever been somal deletional nature of many minisatellite rearrange- performed where the interacting sequences are di- ments. Noncrossover interhomologue events may also verged and might provoke such degradation. Loss of arise by single-strand annealing of newly synthesized DNA in the recipient may also come from cleavage of DNA. In Figure 6 (CЈ–EЈ), a model in which the newly unpaired 3Ј single-strand tails (Figure 6, D and DЈ). synthesized DNA is processively unwound and anneals Such a reaction predicts a role for the Rad1p/Rad10p/ with the other 3Ј single-strand tail is illustrated. Such a Msh2p/Msh3p complex in removing the nonhomolo- mechanism can easily account for array expansions by gous 3Ј tail (Fishman-Lobell and Haber 1992; Sapar- insertion of sequences from one homologue into the baev et al. 1996; Paˆques and Haber 1997; Sugawara other. Finally, a conventional double Holliday junction et al. 1997) as has been demonstrated for mitotic recom- structure (Collins and Newlon 1994; Schwacha and bination. Alternatively, the recently described Rad1p/ Kleckner 1994, 1995) easily accounts for the 31 gene Rad10p independent pathway for removing nonhomol- 18 A. J. R. Bishop, E. J. Louis and R. H. Borts ogous tails may also be acting (Colaiacovo et al. 1999). Buard, J., and G. Vergnaud, 1994 Complex recombination events Ј at the hypermutable minisatellite CEB1 (D2S90). EMBO J. 13: A mechanism not requiring 3 end degradation that 3203–3210. also leads to sequence loss is cleavage of single-strand Cao, L., E. Alani and N. Kleckner, 1990 A pathway for generation loops (Figure 6F) contained within heteroduplex DNA, and processing of double-strand breaks during meiotic recombination in S. cerevisiae. Cell 61: 1089–1101. as envisaged by Fogel et al. (1984) to explain copy Chambers, S. R., N. Hunter, E. J. Louis and R. H. Borts, 1996 number changes at the CUP1 locus. The mismatch repair system reduces meiotic homeologous re- In conclusion, changes in minisatellite size during combination and stimulates recombination-dependent chromosome loss. Mol. Cell. Biol. 16: 6110–6120. meiosis have been characterized at the sequence level. Chen, W., and S. Jinks-Robertson, 1999 The role of the mismatch On the basis of our results we propose that degradation repair machinery in regulating mitotic and meiotic recombina- of the “recipient” DNA molecule occurs during gene tion between diverged sequences in yeast. Genetics 151: 1299– 1313. conversion events and is not readily detected by the Colaiacovo, M. P., F. Paques and J. E. Haber, 1999 Removal of systems normally employed to investigate the mecha- one nonhomologous DNA end during gene conversion by a nisms of recombination. In addition, we propose that RAD1- and MSH2-independent pathway. Genetics 151: 1409– 1423. the heteroduplex formed between newly synthesized Collins, I., and C. S. Newlon, 1994 Meiosis-specific formation of and the recipient DNA can form by single strands of joint DNA molecules containing sequences from homologous DNA annealing in regions of homology. Another inter- chromosomes. Cell 76: 65–75. Crouse, G. F., 1998 Mismatch repair systems in Saccharomyces cerevis- esting finding of this work is that there are recombina- iae, pp. 411–448 in DNA Damage and Repair: Biochemistry, Genetics tion events involving the minisatellite inserts that do and Cell Biology, edited by M. F. Heokstra and J. A. Nickoloff. not result in any change to a novel size. Such events are Humana Press, Totowa, NY. Debrauwere, H., J. Buard, J. 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