The Saccharomyces cerevisiae RDN1 Locus Is Sequestered From Interchromosomal Meiotic Ectopic Recombination in a SIR2-Dependent Manner

Corresponding author: Edward S. Davis, Laboratory of Biochemistry and Genetics, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bldg. 8, Rm. 323, 8 Center Dr. MSC 0840, Bethesda, MD 20892-0840., edwardda{at}intra.niddk.nih.gov (E-mail)

Communicating editor: M. LICHTEN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Meiotic ectopic recombination occurs at similar frequencies among many sites in the yeast genome, suggesting that all loci are similarly accessible to homology searching. In contrast, we found that his3 sequences integrated in the RDN1 (rDNA) locus were unusually poor participants in meiotic recombination with his3 sequences at other sites. We show that the low rate of meiotic ectopic recombination resulted from the poor ability of RDN1::his3 to act as a donor sequence. SIR2 partially repressed interchromosomal meiotic ectopic recombination at RDN1, consistent with its role in regulating recombination, gene expression, and retrotransposition within RDN1. We propose that RDN1 is physically sequestered from meiotic homology searching mechanisms.

DURING meiosis, homologous regions of chromosomes undergo a poorly understood mechanism involving a homology search in order to undergo recombination and proper disjunction. The timing of meiotic recombination events is approximately coincident with chromosome pairing. Double-strand DNA breaks (DSBs), the presumed initiating lesions for meiotic recombination (NICOLAS et al. 1989 ; CAO et al. 1990 ; WU and LICHTEN 1994 ; KEENEY et al. 1997 ), appear before visible synaptonemal complex (SC), while mature recombination structures are detectable near the time of SC breakdown (PADMORE et al. 1991 ).

Most meiotic recombination is allelic (between sites at the same chromosomal position). Eukaryotic genomes contain repetitive DNA in both tandem and dispersed arrangements, creating the potential for ectopic recombination between nonallelic loci. Ectopic recombination includes three classes of events: (1) intrachromosomal (between two different sites on the same chromosome); (2) interhomolog (between two different sites on homologous chromosomes); and (3) interheterolog (between sites on nonhomologous chromosomes). All classes of ectopic recombination have been detected in vegetative and meiotic Saccharomyces cerevisiae cells (KLEIN and PETES 1981 ; JINKS-ROBERTSON and PETES 1985 ; LICHTEN et al. 1987 ; HABER et al. 1991 ; GOLDMAN and LICHTEN 1996 ).

Ectopic recombination occurs at high levels in S. cerevisiae meiosis, where frequencies are generally only 2- to 17-fold lower than that of allelic recombination (JINKS-ROBERTSON and PETES 1985 , JINKS-ROBERTSON and PETES 1986 ; LICHTEN et al. 1987 ; HABER et al. 1991 ; GOLDMAN and LICHTEN 1996 ). Further, the frequencies of ectopic recombination at several sites examined, in which the position of one sequence is held constant, vary over a narrow (2- to 10-fold) range (HABER et al. 1991 ; GOLDMAN and LICHTEN 1996 ). These observations are consistent with a proposal that an initiating DSB on one chromosome is sufficient, or rate limiting, to allow meiotic recombination between homologous sequences at any two given locations (HABER et al. 1991 ). In this view, meiotic recombination is driven by an efficient, yet poorly understood, genome-wide homology search mechanism, creating competition between allelic, sister, and ectopic chromosomal interactions (HABER et al. 1991 ).

Ectopic recombination is potentially hazardous in the presence of dispersed repeated sequences. Ectopic crossing over causes chromosomal abnormalities, including deletions, translocations, and acentric and dicentric chromosomes (JINKS-ROBERTSON and PETES 1986 ; GOLDMAN and LICHTEN 1996 ). Thus, eukaryotes might have evolved mechanisms to avoid ectopic recombination in meiosis. Interhomolog recombination is favored in yeast meiosis by three- to sixfold over intersister recombination (HABER et al. 1984 ; JACKSON and FINK 1985 ; SCHWACHA and KLECKNER 1997 ). Mutations in several genes, including RED1, RAD17, RAD24, MEK1, MEC3, DMC1, UBR1, INP52, BUD3, PET122, ELA1, RAD51, RAD55, and RAD57, reduce interactions between homologs and/or elevate the frequency of unequal sister chromatid exchange (SCHWACHA and KLECKNER 1997 ; THOMPSON and STAHL 1999 ), suggesting that a distinct meiotic machinery exists for promoting allelic recombination.

While ectopic recombination occurs frequently, recombination between heterologous chromosomes is still significantly less efficient than allelic recombination (GOLDMAN and LICHTEN 1996 ). Further, the efficiency of ectopic recombination between sites on homologous chromosomes decreases with increasing distance between the recombination substrates examined. Thus, some aspect of the pairing and alignment of homologous chromosomes favors allelic over ectopic recombination. Meiotic ectopic recombination between subtelomeric regions occurs with approximately twofold greater efficiency than between subtelomeric and interstitial regions (GOLDMAN and LICHTEN 1996 ), consistent with demonstrations of the co-localization of telomeres at the periphery of the nucleus (KLEIN et al. 1992 ). Further, mutations in the checkpoint genes MEC1, RAD17, and RAD24 exhibit elevated intrachromosomal ectopic recombination in meiosis (GRUSHCOW et al. 1999 ), suggesting that the repair of meiotic DSBs is biased toward allelic interactions.

We describe another mechanism limiting the tendency of heterologous chromosomes to undergo ectopic recombination. We developed an assay system that surveys the S. cerevisiae genome for sites with high and low frequencies of meiotic ectopic recombination. We examined interchromosomal meiotic ectopic recombination between two different versions of the yeast his3 gene, one of which was placed at different genomic positions. Though most ectopic pairs behaved as expected, the frequency of ectopic recombination involving the yeast ribosomal DNA (RDN1) locus was ~100-fold lower than at other sites.

RDN1 undergoes very low levels of interhomolog recombination in meiosis (PETES and BOTSTEIN 1977 ). Such behavior could be due simply to a lack of initiating DSBs, while still retaining the ability to act as an efficient recombination donor. However, we present genetic evidence that his3 in RDN1 behaved as an exceptionally poor donor locus in ectopic recombination. We propose that RDN1 is inaccessible, or sequestered, from a homology search mechanism in meiosis.

The silencing gene SIR2 acts at RDN1 by repressing intra- and interhomolog recombination (GOTTLIEB and ESPOSITO 1989 ; SAN-SEGUNDO and ROEDER 1999 ) and RNA polymerase II-mediated transcription (BRYK et al. 1997 ; FRITZE et al. 1997 ; SMITH and BOEKE 1997 ). We observed that a sir2 mutation partially elevated the rate of ectopic recombination involving RDN1, but not at other sites. Silencing mechanisms play a partial role in controlling the accessibility of donor sequences in ectopic recombination.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast strains, media, and genetic manipulations:
The yeast strains used in this study are described in Table 1. Genomic DNA isolation was performed as described (ROSE et al. 1990 ). Synthetic medium in plating experiments, except for that summarized in Table 2, was synthetic minimal medium (ROSE et al. 1990 ). For the experiment summarized in Table 2, AA, or synthetic complete medium (ROSE et al. 1990 ) containing 86 mg liter^-1 adenine and 17 mg liter^-1 para-amino benzoic acid was used. Yeast transformations were carried out by the lithium acetate procedure (GIETZ et al. 1992 ) or the glusulase-spheroplast procedure (HINNEN et al. 1978 ). Mating type and mating proficiency were determined by mating to tester strains DC14 and DC17. Disruption of SIR2 was achieved by transforming haploid strains with plasmid pBSTsir2::URA3 (kindly provided by M. Bryk), which carries the hisG-URA3-hisG cassette (ALANI et al. 1987 ) inserted in the BglII site at nucleotide +823 of the protein-coding region. SIR2 disruptions were confirmed by the inability of cells to mate and by Southern blotting analysis. Homozygous sir2 disruption diploid strains were constructed by polyethylene glycol (PEG) fusion in which haploid cells were prepared by the glusulase-spheroplast procedure for DNA transformation and mixed in the presence of polyethylene glycol (PEG 3350). Mixtures were plated on synthetic medium that selects for the diploids.

Recombination substrates:
The Ty1mhis3-AI (CURCIO and GARFINKEL 1991 ), trp1-089-his3-621 (MCGILL et al. 1990 ), and his3-MscI (DERR and STRATHERN 1993 ) constructs are described in previous studies. The trp1-089-his3-621 and his3-MscI cassettes were inserted at an EcoRI site on chromosome III near MATa at nucleotide C196972, using the numbering system of the Saccharomyces Genome Database (http://genome-www.stanford.edu/Saccharomyces). Single unspliced (His^-) Ty1mhis3-AI insertions were previously generated in haploid strains (BRYK et al. 1997 ; M. J. CURCIO, personal communication). The Ty1mhis3-AI insertion site junctions in strains JC234, JC242, JC272, and JC815 (haploid parents of ED291B, ED292A, ED293A, and ED295A, respectively) were determined by BRYK et al. 1997 . The Ty1mhis3-AI insertion site junctions in strains DG1252, DG1348, and JC227 (haploid parents of ED288A, ED289A, and ED290A, respectively) were determined in this study by recovering plasmids from His⁺, MAT meiotic segregants of the diploids in Escherichia coli strain KC8 (hsdR leuB600 trpC9830 pyrF::tn5 hisB463 lacD X74 strA galU,K; kindly provided by K. Struhl via M. Mastrangelo). S. cerevisiae HIS3 complements the hisB463 mutation in E. coli (STRUHL et al. 1976 ); therefore, ampicillin-resistant colonies were replica plated to medium that selects for histidine prototrophy. The Ty1/yeast and yeast/plasmid junctions of recovered His⁺ plasmids were sequenced using the ABI Prism DNA Sequencing kit (PE-Applied Biosciences, Inc.). The Ty1mhis3-AI 5' LTR/yeast junction sequence in JC815 (haploid parent of ED295A) was also determined in this fashion, confirming the results of BRYK et al. 1997 . The Ty1mhis3-AI insertion site junction in strain JC273 (haploid parent of ED294A) was determined by vectorette polymerase chain reaction (PCR; RILEY et al. 1990 ) on isolated DNA, followed by DNA sequencing.

The TRP1-his3-621 DNA cassette was constructed as follows: PCR products of TRP1 (nucleotides -102 to +755) and HIS3 (nucleotides -189 to +819) were synthesized and cloned as ApaI-XhoI fragments in pBluescript II KS(+) and pBluescript II SK(-) (Stratagene, La Jolla, CA), respectively. Both constructs were digested with ApaI and ScaI and joined together. The new plasmid, pESD62, has TRP1 and HIS3 in divergent transcriptional orientations, with the pBluescript II multiple cloning site polylinker from SacI to XhoI in a palindromic repeat flanking the cassette. The Asp718 fill-in mutation of his3-621 was introduced by replacing the NsiI-NdeI fragment of HIS3 with the corresponding region from cloned his3-621, generating pESD64.

Fragments from RDN1 (with respect to the start of the 35S rRNA gene: nucleotides -1078 to -471 with ApaI and XhoI ends; nucleotides -470 to +200 with XhoI and SacI ends) and CUP1 (nucleotides -818 to +50 with ApaI and XhoI ends; nucleotides +51 to +463 with XhoI and SacI ends) were synthesized by PCR. PCR products were digested with the appropriate restriction enzymes and joined at their XhoI ends by cloning into pBluescript II SK(-). TRP1-his3-621 was liberated from pESD64 with XhoI and cloned into the XhoI site of the RDN1 and CUP1 plasmids in both orientations. The TRP1-his3-621 insertion site within an RDN1 repeat unit in ED403-18 and ED404-19 is identical to that of Ty1mhis3-AI in ED293A (BRYK et al. 1997 ). For insertion into ARG4, a plasmid containing the ARG4 gene (kindly provided by M. Lichten) was digested with AccI (nucleotide +230) and made blunt ended using Klenow enzyme. TRP1-his3-621 was liberated from pESD64 with SmaI and ligated to the filled-in AccI site of ARG4 in both orientations. All TRP1-his3-621 insertions were generated by transformation and selection for a Trp⁺ phenotype in haploid strains. The location and orientation of TRP1-his3-621 insertions in vivo was confirmed by Southern blot analysis.

Recombination measurements:
Three single colonies of each diploid yeast strain were cultured at 30° in 200 ml liquid YEP (1% yeast extract, 2% Bacto-peptone) containing 2% potassium acetate to a density of ~5 x 10⁶ cells ml^-1. Just immediately prior to sporulation, ~1 x 10⁸ cells were removed for DNA preparation to be used in Southern blotting analysis. To determine the frequencies of vegetative His⁺, Ura⁺, and Leu⁺ events, one-half of the remaining culture was pelleted and resuspended in H₂O. Dilutions were plated on YEP containing 2% glucose (YEPD) and synthetic medium lacking either histidine, uracil, or leucine. Frequencies were calculated by dividing the number of prototrophs on selective plates by the number of colonies on YEPD. The other half of the culture was pelleted, resuspended at a density of ~5 x 10⁷ cells ml^-1 in minimal sporulation medium (2% potassium acetate, 2.5 mg liter^-1 phenylanine, 1 mg liter^-1 each adenine sulfate and uracil, 0.5 mg liter^-1 each histidine-hydrochloride, leucine, lysine-hydrochloride, tryptophan, methionine, and arginine, 0.4 mg liter^-1 tyrosine, and 0.2 mg liter^-1 proline), and agitated at 30° for 3 days, generating spore-containing asci.

Spores were liberated from asci as follows: Incubation in 5.0 ml 2.5% glusulase at 30° for 1 hr, followed by addition of 5.0 ml ice-cold 0.5% Triton/10 mM EDTA. Asci were pelleted by centrifugation, washed three times in 10 ml ice-cold 0.5% Triton/10 mM EDTA, and resuspended in 5.0 ml ice-cold 0.5% Triton/10 mM EDTA. Asci were sonicated on ice using a Vibra-Cell sonicator (Sonics and Materials, Inc.; 80% duty cycle, microprobe output setting ~4.5) 3 x 40 sec, pelleted by centrifugation, and washed twice more in 5.0 ml ice-cold 0.5% Triton/10 mM EDTA. Spores were resuspended in 1.0 ml ice-cold 0.5% Triton/10 mM EDTA. Spore concentrations were adjusted to ~1 x 10⁸ ml^-1. The fraction of single spores following this procedure was ~75%. Spores were diluted appropriately in 0.5% Triton/10 mM EDTA and plated on YEPD and synthetic medium lacking histidine, uracil, or leucine. Frequencies of meiotic recombination to His⁺, Ura⁺, and Leu⁺ were determined by dividing the number of colonies arising on selective media by the number of haploids, as estimated by doubling the number of red colonies on YEPD plates derived from the heterozygous ade2 marker. The fraction of red colonies on YEPD plates ranged from 42 to 54%, indicating that most colonies arising after the isolation procedure were derived from spores.

Donor/recipient ratios:
A total of 100 His⁺ recombinants from each diploid strain following sporulation were patched to AA-his plates. After overnight growth, patches were replica plated to mating-type testers and to various media to determine genetic markers. All cryptopleurine-sensitive (CRY1) MAT His⁺ recombinants were considered to have their His⁺ phenotype unlinked to MAT, because such recombinants should occur via nonreciprocal gene conversion of Ty1mhis3-AI. MATa His⁺ spore progeny were replica plated to plates containing spreads of strain ED315-105c (relevant genotype MAT ura3-52 his3-200) and permitted to mate. Diploid patches were selected on synthetic minimal medium lacking histidine, leucine, adenine, tryptophan, and lysine. These patches were sporulated and replica plated to strains GRY1580 and GRY1582 (relevant genotypes MATa and MAT, respectively, URA3 his3-200) on YEPD. After overnight incubation allowing mating, the plates were replica plated to synthetic complete medium lacking histidine and uracil. Under these conditions, if His⁺ was linked to MATa, then second-generation diploid patches would grow only when spores mated to GRY1580 (MAT). If the His⁺ phenotype was not linked to MAT, patches would grow with similar efficiencies when spores mated with GRY1580 (MAT) or GRY1582 (MATa). A similar test to confirm that the HIS3 recombinant gene in MAT cry1 His⁺ segregants was linked to MAT was performed by mating to the MATa ura3-52 his3-200 strain ED315-93b.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The S. cerevisiae genome contains sites that undergo high ("hotspots") and low ("coldspots") levels of meiotic recombination. Many chromosomal sites that sustain high frequencies of meiotic DSBs undergo high frequencies of meiotic recombination (SUN et al. 1989 ; WU and LICHTEN 1994 , WU and LICHTEN 1995 ; BAUDAT and NICOLAS 1997 ; BORDE et al. 1999 ). However, only a small number of hotspots have been characterized in detail (NICOLAS et al. 1989 ; MALONE et al. 1994 ; FAN et al. 1995 ; LICHTEN and GOLDMAN 1995 ). We were interested in making a genome-wide survey for additional hotspots and coldspots to better understand how the effect of genome position can govern the efficiency of meiotic recombination.

To identify additional hotspots and coldspots of meiotic recombination, we designed a system comparing the frequencies of meiotic recombination between a pair of homologous recombination substrates placed in several ectopic positions (Fig 1). A bank of isogenic diploid strains was generated, each carrying two different mutant his3 genes. All strains carried one mutant his3 at a constant position, near MATa, and a second mutant his3, the variable, at distinct ectopic sites. Meiotic DSBs, the presumed initiating lesions for meiotic recombination in S. cerevisiae, occur at normal levels on chromosomes even in the absence of nonsister homology (NICOLAS et al. 1989 ; GILBERTSON and STAHL 1994 ; WU and LICHTEN 1995 ). Therefore, we did not expect that varying the position of one recombination substrate would significantly affect the ability of the constant substrate to sustain initiation events. Thus, in principle, any differences in meiotic recombination rates among the various ectopic pairs were expected to reflect changes in the properties of the variable recombination substrate when moved to different locations.

View larger version (32K): In this window In a new window Download PPT slide   Figure 1. Strategy for identifying genomic sites with exceptional properties in meiotic recombination. (Top) A nonfunctional his3 gene (his3-a) is introduced into a haploid strain in which the resident HIS3 was deleted. Individual derivatives carrying his3-a at different sites are isolated and mated to another haploid carrying a second mutant his3, his3-b, placed near MATa. The resulting isogenic diploids (bottom) are unable to grow on medium lacking histidine (His-). Diploids are sporulated and allowed to complete meiosis. Ectopic recombination between his3-a and his3-b can create a fully functional HIS3 gene, permitting the cell to grow on synthetic medium lacking histidine (His+).

We used variable his3 insertions generated by two different methods. In one set of experiments (Fig 2A), his3-AI was placed in variable positions by the S. cerevisiae retrotransposon Ty1 (BRYK et al. 1997 ; M. J. CURCIO, personal communication). Haploid strains previously found to carry single, unspliced (His^-) Ty1mhis3-AI insertions were chosen. Insertion sites were determined subsequently. Each Ty1mhis3-AI-bearing haploid was mated to a second strain, GRY818, carrying his3-621 inserted in constant position on chromosome III between CRY1 and MATa. In the second set of experiments (Fig 2B), his3-621 was inserted at variable positions in haploids by targeted integration using selection for a linked TRP1 gene. TRP1-his3-621 haploids were mated to a second strain, ED361A-1, carrying his3-MscI near MATa (same position and orientation as his3-621 in GRY818). Recombination between the his3 markers at variable loci and MATa::his3 can generate His⁺ colonies, which are visualized in patches or quantitated among random spores. The MAT-CRY1 interval was chosen for integration of the constant his3 because, being a known coldspot for meiotic recombination (OLIVER et al. 1992 ; BAUDAT and NICOLAS 1997 ), it was expected to maximize the sensitivity of detecting insertion sites that are hotspots for meiotic recombination.

View larger version (33K): In this window In a new window Download PPT slide   Figure 2. his3 recombination substrates. (A) Ty1mhis3AI x MATa::trp1-089-his3-621. Ty1mhis3-AI (variable) is a hybrid Ty1 transposable element (white) carrying a defective his3 gene (gray) inserted near the 3' long-terminal repeat (boxes with arrows). his3-AI contains a 104-bp artificial intron (AI) inserted at the MscI site. (B) TRP1-his3-621 x MATa::his3MscI. TRP1-his3-621 (variable) is his3-621 (gray), a defective his3 gene containing a fill-in of the Asp718 site, linked to a fully functional TRP1 gene (crosshatched) in divergent transcriptional orientation. TRP1-his3-621 is flanked by a partial pBluescript II polylinker (Stratagene) in a palindromic repeat. Large arrows represent the direction of transcription. Small arrows represent the direction of the polylinker from the SacI site to the XhoI site. TRP1-his3-621 can be inserted at any locus in the S. cerevisiae genome by standard cloning and transformation techniques and selection for the ability to grow on synthetic medium lacking tryptophan.

Each diploid also carried the ura3-52 and ura3-167 mutations in allelic positions at URA3 on chromosome V. Recombination between these can generate Ura⁺ spores, which we quantitated as a control for sporulation and overall recombination efficiency.

Initial test of the ectopic recombination system:
As described below, we observed efficient meiotic ectopic recombination between the his3-AI sequences inserted at several sites by Ty1 and the his3-621 gene near MATa. However, the identification of the His⁺ prototrophs required verification that those colonies arose from meiotic recombination and not by retrotransposition or RNA-mediated recombination.

The increase in His⁺ frequencies upon sporulation is illustrated by the behavior of two diploid strains, ED292A (carrying a Ty1mhis3-AI on chromosome XII at position L1060532, using the numbering system of the Saccharomyces Genome Database) and ED294A (carrying a Ty1mhis3-AI on chromosome II at position B326918). The mitotic levels of His⁺ for ED292A and ED294A were 1.0 x 10^-7 and 1.3 x 10^-7, respectively. Upon sporulation the His⁺ frequencies increased over 100-fold to 3.1 x 10^-5 and 5.3 x 10^-5, respectively. An internal control for allelic recombination using mutations in ura3 showed a 100-fold increase upon sporulation over mitotic levels (from 1.8 x 10^-5 to ~2 x 10^-3). This increase in His⁺ frequency is consistent with meiotic ectopic recombination between the Ty1mhis3-AI element and the his3-621 sequence at MAT. However, Ty1mhis3-AI can generate His⁺ cells via Ty1 transposition (CURCIO and GARFINKEL 1991 ) or RNA-mediated gene conversion (DERR et al. 1991 ). To determine the contribution of these mechanisms to the frequencies of meiotic His⁺ events in our ectopic recombination system, we replaced his3-621 near MATa in ED292A and ED294A with his3-MscI. The his3-AI and his3-MscI mutations are at the same position in HIS3 and hence cannot recombine to generate HIS3. A His⁺ phenotype can arise in the his3-MscI x Ty1mhis3-AI diploids only via Ty1mhis3-AI transposition to a new site, or RNA-mediated gene conversion between Ty1mHIS3 cDNA and endogenous Ty1 elements or the MATa::his3-MscI sequence.

The frequencies of meiotic His⁺ formation in the MATa::his3-MscI x Ty1mhis3-AI derivatives of ED292A and ED294A (ED308A and ED310B, respectively) following sporulation were 1 x 10^-7 and 0.2 x 10^-7, about the same as their mitotic levels. Thus meiosis did not strongly increase the level of Ty1mhis3-AI transposition. In summary, these results support the conclusion that virtually all His⁺ events observed following sporulation of MATa::his3-621 x Ty1mhis3-AI diploids were due to meiotic ectopic recombination.

RDN1 is a poor participant in meiotic ectopic recombination:
We determined whether placing Ty1m his3-AI in different locations affected the frequency of meiotic ectopic recombination. Eight diploids were examined, each containing his3-621 near MATa and Ty1mhis3-AI in different places. Six strains (ED288A, ED289A, ED290B, ED292A, ED294A, and ED295A; Table 2) exhibited a frequency of meiotic recombination to His⁺ varying over only a sixfold range. A similar range of meiotic recombination frequencies among different ectopic crosses, in which the position of one sequence is held constant, also has been observed previously (GOLDMAN and LICHTEN 1996 ).

Two strains, ED291B and ED293A (Table 2), behaved much differently from the other six. The frequencies of meiotic His⁺ events in ED291B and ED293A were ~100-fold lower than that of the other six strains of Table 2. In all of the strains of this experiment, the frequencies of allelic recombination at the control locus, URA3, varied over less than a twofold range. Thus, the low frequency of meiotic His⁺ formation in ED291B and ED293A was not due to a defect in sporulation or the general machinery of meiotic recombination, but rather to the effect of genomic position on the Ty1mhis3-AI recombination substrate. Interestingly, both ED291B and ED293A contained Ty1mhis3-AI insertions in the nontranscribed spacer region of the ribosomal DNA (RDN1), a cluster of 100–200 9.1-kb transcription units arranged in a direct repeat on chromosome XII (PETES and BOTSTEIN 1977 ).

Although meiotic allelic recombination at RDN1 occurs at very low frequency (PETES and BOTSTEIN 1977 ), the result presented in Table 2 was unexpected, given the proposal that homology searching makes all genomic sites accessible to ectopic recombination. This result suggests that events that initiated at the constant his3 sequence and that were sufficient to promote recombination with his3 at most of the ectopic sites we tested were not sufficient to promote recombination with a his3 insert within RDN1. Therefore, we considered several possibilities to account for our observation of such an exceptionally low rate of meiotic ectopic recombination involving RDN1.

First, we eliminated the possibility that the Ty1mhis3-AI sequences in these strains might have been lost before meiosis due to recombination between the tandemly repeated RDN1 units. In the experiment summarized in Table 2, DNA was extracted just before meiosis from one sample each of diploids ED292A (carrying Ty1mhis3AI on chromosome XII, outside RDN1) and ED294A (carrying Ty1mhis3-AI on chromosome II); three samples each of the diploids carrying Ty1mhis3-AI inserts in RDN1 (ED291B and ED293A); one sample each of the haploids JC234, JC242, JC272, and JC273 (parents of ED291B, ED292A, ED293A, and ED294A, respectively); and the other haploid parent, GRY818 (no Ty1mhis3-AI insert; his3-621 near MATa). DNA was digested with ClaI, Southern blotted, and probed with his3 DNA. We observed that the intensity of the 1026-bp his3-AI fragment was similar among the eight diploid samples (data not shown), indicating that the low rate of meiotic ectopic recombination involving RDN1 was not due to large-scale loss of Ty1mhis3-AI prior to meiosis.

In similar fashion, we asked whether Ty1mhis3-AI might have been lost from RDN1 during meiosis, possibly due to unequal sister chromatid recombination. We isolated 44 haploid colonies from ED293A following meiosis from the nonselective (YEPD) plates in the experiment summarized in Table 5. Because the Ty1mhis3-AI insert is hemizygous in ED293A, a well-maintained insert should be present in ~22 (50%) of the haploid meiotic derivatives. DNA was isolated from each haploid, digested with ClaI, Southern blotted, and probed with his3 DNA. We found that 26 of the 44 haploids (59%) still retained the 1026-bp his3-AI insert in RDN1 following meiosis (data not shown). This result indicates that Ty1mhis3-AI is well maintained in RDN1, yet is nearly unable to participate in meiotic ectopic recombination.

View this table: In this window In a new window   Table 3. TRP1-his3-621 x MATa::his3-MscI

The low rate of ectopic recombination at RDN1 is independent of Ty1:
Another possible explanation for the low rate of meiotic ectopic recombination involving RDN1 is that the presence of Ty1 sequences in ED291B and ED293A might have created a substrate that was a poor participant in ectopic recombination at RDN1, since these insertions are sensitive to rDNA silencing (BRYK et al. 1997 ). A similar phenomenon was also reported by SMITH and BOEKE 1997 , although they also found that a minimal URA3 promoter is silenced at RDN1 without Ty1 sequences. To determine whether other substrates without Ty1 sequences were also poor participants in meiotic ectopic recombination when inserted at RDN1, we made a new recombination substrate. This construct contained his3-621, marked with TRP1 and lacking Ty1 DNA (Fig 2B). TRP1-his3-621 can recombine with his3-MscI (placed in the constant position in the MATa-CRY1 interval) to yield His⁺ prototrophs.

We placed TRP1-his3-621 in diploids at the ARG4, RDN1, and CUP1 loci. The cassette was inserted in both possible orientations, to determine any potential influence of ectopic crossovers that lead to acentric and dicentric chromosomes. In addition to measuring allelic recombination at URA3 (ura3-52 x ura3-167), we included an internal control for meiotic ectopic recombination to Leu⁺ (leu2-K x lys2::leu2-R).

When TRP1-his3-621 was inserted at ARG4, we observed efficient meiotic ectopic recombination to His⁺ (Table 3). The frequency of meiotic recombination was about 100-fold higher than during vegetative growth (data not shown). Orientation of the cassette did not significantly alter the frequency of meiotic ectopic recombination to His⁺ (Table 3, compare ED413-1 with ED414-1).

When TRP1-his3-621 was inserted at RDN1, the frequency of meiotic ectopic recombination to His⁺ was about 100-fold lower than in the strain carrying the variable substrate at ARG4 (Table 3). Orientation of the cassette did not significantly affect the frequency of ectopic recombination to His⁺ (Table 3; compare ED403-18 with ED404-19). Meiotic allelic recombination at the control URA3 locus varied by less than a factor of two among the strains carrying TRP1-his3-621 at ARG4 or RDN1. Therefore, the low rate of meiotic ectopic recombination involving RDN1::TRP1-his3-621 was not due to an inability to induce meiotic recombination.

We also determined whether the low rate of meiotic ectopic recombination involving RDN1 was a common property of naturally occurring direct repeats. We inserted TRP1-his3-621 at the CUP1 locus on chromosome VIII. CUP1 is a 2.1-kb gene that, like RDN1, exists in a 2- to 30-copy tandemly repeated array (FOGEL and WELCH 1982 ). Our strains carried at least three repeat units, as estimated by Southern blotting (E. S. DAVIS and J. N. STRATHERN, unpublished data). Although the position of insertion of TRP1-his3-621 within a CUP1 repeat unit was confirmed by Southern analysis, the repeat unit within the array carrying the insertion was not identified. The frequency of ectopic recombination at CUP1 was about 2.5-fold lower than at ARG4 and at least 35-fold higher than at RDN1. As for ARG4 and RDN1, orientation of the TRP1-his3-621 cassette did not significantly influence the frequency of meiotic ectopic recombination to His⁺ (Table 3; compare ED 409-1 with ED410-1).

These results permit the following conclusions: (1) The low rate of meiotic ectopic recombination between MATa::his3 and RDN1::his3 did not require the presence of Ty1 sequences; (2) the low rate of meiotic ectopic recombination involving RDN1 was independent of the orientation in which TRP1-his3-621 was inserted; (3) TRP1-his3-621 inserted within ARG4 underwent efficient meiotic ectopic recombination; and (4) despite being a directly repeated locus itself, CUP1 was also an efficient participant in meiotic ectopic recombination.

RDN1 is a poor donor locus in meiotic recombination:
One published report provides evidence that meiotic DSBs that initiate recombination do not occur within RDN1 (HOGSET and OYEN 1984 ). Therefore, another explanation for the low rate of meiotic ectopic recombination involving RDN1 is that the constant MATa::his3 was also unable to initiate meiotic ectopic recombination. Two experiments were conducted to address this possibility.

In the first experiment, we estimated the rate of initiation of meiotic ectopic recombination near MATa::his3-621. In meiotic gene conversion in S. cerevisiae, the chromosome sustaining DSBs tends to act as the recipient (NICOLAS et al. 1989 ). We have observed a similar bias of the initiating chromosome acting as recipient in mitotic recombination events (MCGILL et al. 1993 ). Therefore, the frequency at which MATa::his3-621 became His⁺ should reflect the rate of initiation of meiotic ectopic recombination at that locus. As the his3-621 sequence was tightly linked to MATa in the strains of Table 2, we determined the percentage of His⁺ events linked to mating type among spore progeny in the six strains with Ty1mhis3-AI insertions outside RDN1.

Among the six strains with Ty1mhis3-AI insertions outside RDN1, the fraction of His⁺ events linked to MATa ranged from 26 to 90% (Table 2). These data suggest that MATa::his3-621 was about as likely to serve as a recipient of His⁺ information in meiotic recombination as the Ty1mhis3-AI insertions outside RDN1. To estimate the frequency at which MATa::his3-621 serves as recipient in the six strains with Ty1mhis3-AI insertion sites outside RDN1, we multiplied the percentage of His⁺ events linked to MATa by the overall rate of meiotic ectopic recombination to His⁺. These calculations resulted in frequencies that ranged from 0.63 x 10^-5 to 2 x 10^-5. The overall rate of meiotic ectopic recombination in the strains carrying Ty1mhis3-AI at RDN1 was much lower than this estimated initiation rate, suggesting that his3 inserted at RDN1 was a poor donor in meiotic ectopic recombination with his3 inserted near MATa.

The MAT-CRY1 interval on chromosome III is a coldspot for meiotic recombination (OLIVER et al. 1992 ), sustaining relatively few meiotic DSBs (BAUDAT and NICOLAS 1997 ). We hypothesized that, if RDN1::his3 was a poor donor with MATa::his3, then RDN1::his3 also should be a poor donor with a locus that undergoes efficient ectopic recombination with MAT. We conducted a second experiment, in which we made ARG4 the site of the constant his3 substrate, TRP1-his3-621, instead of the trp1-089-his3-621 insertion near MATa. We constructed a set of such diploid strains, ED491A, ED503C, ED485B, and ED497A, which were isogenic with ED292A, ED294A, ED291B, and ED293A, respectively, from the experiment summarized in Table 2. However, we note that while the natural ARG4 locus is a hotspot for allelic recombination in meiosis (NICOLAS et al. 1989 ), we do not know whether DSBs characteristic of ARG4 hotspot activity are occurring in the arg4::TRP1-his3-621 construct or whether such DSBs outside the region of homology will be sufficient to initiate ectopic recombination.

We compared the frequencies of meiotic ectopic recombination between arg4::TRP1-his3-621 and the Ty1mhis3-AI insertions at different sites within this new strain set. A result similar to that presented in Table 2 was obtained (Table 4). Strains containing Ty1mhis3-AI insertions at RDN1 were still two orders of magnitude less proficient in meiotic ectopic recombination to His⁺ than strains with Ty1mhis3-AI insertions elsewhere. In addition, MATa::his3 and arg4::his3 recombined efficiently with each other (Table 3). Taken together, these results suggest that neither MATa::his3 nor arg4::his3 was capable of efficient recombination with RDN1::his3.

SIR2 participates in the sequestration of RDN1:
Loss of SIR2 function elevates the rate of mitotic and meiotic interhomolog and intrachromosomal recombination at RDN1 (GOTTLIEB and ESPOSITO 1989 ; SAN-SEGUNDO and ROEDER 1999 ). Therefore, we examined the role SIR2 plays in meiotic ectopic interchromosomal recombination involving RDN1 by constructing appropriate diploid strains homozygous for disruption of SIR2 (Table 5). SIR2 disruption elevated the rate of meiotic interchromosomal recombination between MATa::his3-621 and two different Ty1mhis3-AI insertions in RDN1 ~15- to 30-fold (compare ED291B with ED347C-1 and ED293A with ED349G-1). The sir2 disruption did not increase the frequency of ectopic recombination to His⁺ at other loci (compare ED292A with ED348A-2 and ED294A with ED350A-1) or significantly increase the frequency of allelic recombination to Ura⁺.

We also inactivated SIR2 in the strains carrying TRP1-his3-621 at RDN1. In these strains, sir2 disruption caused a reproducible five- to eightfold increase in the frequency of meiotic ectopic recombination involving RDN1 (Table 3; compare ED479-62 and ED480-13 with ED403-18 and ED404-19, respectively). This relative increase was less than the sir2-mediated, 15- to 30-fold elevation of meiotic ectopic recombination involving RDN1 when Ty1mhis3-AI was the substrate (compare Table 3, ED479-62 and ED480-13, with Table 5, ED347C-1 and ED349G-1), although the absolute frequencies were comparable.

We demonstrated that the low level of ectopic recombination did not reflect loss of the TRP1-his3-621 sequences from RDN1 before or during meiosis by scoring the Trp⁺ phenotype in spore clones. Among the Ade^- spore-derived colonies from the strains of Table 3, 60% from ED403-18 (SIR2/SIR2) and 53% from ED479-62 (sir2::hisG/sir2::hisG) were Trp⁺ (~50% were expected to still carry the insert). The presence of a full-length TRP1-his3-621 insert at the onset of sporulation in the SIR2 and sir2::hisG diploids was confirmed by Southern blotting (not shown).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Meiotic ectopic recombination between two different his3 sequences occurred at an exceptionally low rate when one sequence was inserted in the yeast rDNA array (RDN1). Two lines of evidence suggest that RDN1 acted as an unusually poor donor sequence in meiotic ectopic recombination.

Meiotic ectopic recombination between MATa::his3 and RDN1::his3 occurred at low frequency, despite evidence that MATa::his3 acted as an efficient recipient of His⁺ events and presumably was sustaining initiating chromosome breaks (Table 2). The frequency at which MATa::his3-621 served as recipient in the six strains with Ty1mhis3-AI insertion sites outside RDN1 ranged from 0.63 x 10^-5 to 2 x 10^-5. If this range of frequencies reflected the rate of initiation of meiotic ectopic recombination at MATa::his3-621 and if an initiating break on one chromosome is rate limiting, then the frequency of meiotic ectopic recombination between MATa::his3-621 and any other site should fall within this range of ~1 x 10^-5. However, the frequency of meiotic His⁺ formation in the strains carrying Ty1mhis3-AI at RDN1 was about 100-fold lower than this apparent initiation rate (Table 2). This result suggests that despite efficient initiation of meiotic ectopic recombination at MATa::his3-621, RDN1::Ty1mhis3-AI was a poor template for repair of these events.

Second, RDN1::his3 was a poor participant in meiotic ectopic recombination with MATa::his3 or arg4::his3. In contrast, MATa::his3 and arg4::his3 recombined well (1.5 x 10^-4) with each other (Table 3), demonstrating that one or both loci were capable of initiating ectopic recombination in meiosis. If initiation is rate limiting, either MATa::his3 and/or arg4::his3 should undergo efficient ectopic recombination with RDN1::his3. In contrast, the rates of ectopic recombination to His⁺ in the RDN1::his3 x arg4::his3 crosses were not significantly different from those in the RDN1::his3 x MATa::his3 crosses. We argue that the S. cerevisiae RDN1 locus served as a poor donor because it was inaccessible, or sequestered from a genome-wide homology search employed by meiotic cells. This model predicts that an initiating DSB in a his3 outside RDN1, if unable to recombine with RDN1::his3, will instead be repaired using the homolog or the sister chromatid as a template.

Our results demonstrate that the initiating event alone is not sufficient for meiotic ectopic recombination. A previous study suggests that all genomic regions can act as donors with similar propensity (HABER et al. 1991 ). However, those experiments examined a small number of donors, and most of the donors were on the same chromosome as the recipient. Among other ectopic crosses, in which the position of one substrate is held constant, the frequencies of interchromosomal meiotic ectopic recombination vary over rather narrow (2- to 10-fold) ranges (GOLDMAN and LICHTEN 1996 ). As shown by GOLDMAN and LICHTEN 1996 , however, allelic and ectopic recombination between homologs is more efficient than ectopic recombination between heterologous chromosomes, suggesting some role for homologous chromosome pairing in enhancing the efficiency of meiotic recombination.

RDN1 undergoes much less meiotic crossing over than expected from its size (PETES and BOTSTEIN 1977 ; ZAMB and PETES 1982 ), but experiences frequent meiotic unequal sister chromatid exchange (USCE; PETES 1980 ). These observations can be explained by the following scenarios: First, RDN1 lacks initiating DSBs required for crossover (HOGSET and OYEN 1984 ), while USCE occurs by a different mechanism than allelic recombination. Accordingly, mitotic USCE within RDN1 is RAD52 independent (ZAMB and PETES 1981 ). Second, the Hop1p protein, which is required for promoting the formation of meiotic allelic recombination intermediates at the expense of USCE (SCHWACHA and KLECKNER 1994 ), is normally excluded from the nucleolus (SAN-SEGUNDO and ROEDER 1999 ). Mutations in SIR2 and PCH2, however, cause mis-localization of Hop1p to the nucleolus, and RDN1 becomes competent for allelic recombination (SAN-SEGUNDO and ROEDER 1999 ).

A precedent for the idea that not all sequences act as efficient recombination donors has been observed in mating-type interconversion in the yeasts S. cerevisiae and Schizosaccharomyces pombe. The directionality that mating-type interconversion exhibits, that is, the tendency of MAT cells to switch to MATa or mat1-p cells to switch to mat1-m, may reflect a cell-type-dependent sequestration of the donor sequences related to the meiotic sequestration of RDN1 described here.

Mating-type interconversion is a mitotic gene conversion event, initiated by a site-specific chromosome break at the mating-type locus (MAT in S. cerevisiae; mat1 in S. pombe). Both yeasts carry transcriptionally silenced copies of mating-type genes distal to the mating-type loci (HML and HMRa, in S. cerevisiae; mat2-P and mat3-M to the right of mat1 in S. pombe). The mating-type loci are flanked by DNA having perfect homology with both silent cassettes, yet during switching haploid cells are selective, because they use only the donor locus normally carrying information of the opposite mating type. Hence, the accessibility of the ectopic donor sequences is regulated by mating type. Similar to our observation of meiotic recombinational sequestration of RDN1, when MAT is undergoing efficient initiation of recombination stimulated by the Ho-endonuclease, it is unable to participate in ectopic recombination with one of the unlinked, homologous loci.

Cis-acting elements and trans-acting factors regulate the nonrandom donor choice during interconversion, but the precise mechanism seems to be different in both yeasts. In both systems, donor choice is associated with repression of recombination within nearby intervals (THON and KLAR 1993 ; WU and HABER 1996 ; GREWAL and KLAR 1997 ; SZETO et al. 1997 ; SZETO and BROACH 1997 ). In S. pombe, chromatin structure modification is proposed to play a role in regulating donor choice (GREWAL and KLAR 1997 ).

How might the role of Sir2p in meiotic sequestration of RDN1 described here be related to its known roles in silencing and rDNA mitotic stability? SIR2 was first defined on the basis of its requirement in silencing transcription of HML and HMRa (KLAR et al. 1979 ; IVY et al. 1986 ). Transcriptional silencing at telomeres is also Sir2p dependent (APARICIO et al. 1991 ), and silencing of pol II promoters inserted within the rDNA repeat array was recently also shown to be dependent on Sir2p (BRYK et al. 1997 ; SMITH and BOEKE 1997 ). Sir2p has not been shown to have a direct role in the recombination associated with mating-type switching beyond blocking access of the Ho-endonuclease to its potential recognition sites in HML and HMR. In contrast, Sir2p's activity in the rDNA repeats was first revealed by its role in repressing inter- and intrachromosomal recombination at RDN1 (GOTTLIEB and ESPOSITO 1989 ; SAN-SEGUNDO and ROEDER 1999 ). Sir2p does not bind DNA, but has both in vitro ADP-ribosyltransferase (TANNY et al. 1999 ) and NAD-dependent histone deacetylase (SHIN-ICHIRO et al. 2000 ) activities. One or both of these activities appear to be essential for silencing in vivo (TANNY et al. 1999 ; SHIN-ICHIRO et al. 2000 ). In addition, SIR2 overexpression causes histone deacetylation (BRAUNSTEIN et al. 1993 ).

Mitotic and meiotic recombination occur at higher levels in transcriptionally active DNA (THOMAS and ROTHSTEIN 1989 ; WU and LICHTEN 1994 ), although transcription itself is not required for meiotic recombination (SCHULTES and SZOSTAK 1991 ; WHITE et al. 1992 ). These findings suggest that efficient recombination requires the presence of DNA that is accessible to recombination factors. Sir2p probably plays a role in the establishment or maintenance of chromatin structure at RDN1. Recombinational sequestration and RNA polymerase II-dependent transcriptional silencing might share some common chromatin structure determinants. Consistent with this idea, transcription of the RDN1::Ty1mhis3-AI elements in strains ED291B and ED293A is silenced in a SIR2-dependent manner in their haploid parents JC234 and JC272, respectively (BRYK et al. 1997 ).

Our results demonstrate that RDN1 was sequestered from meiotic ectopic recombination in a SIR2-dependent manner whether we used Ty1mhis3-AI or TRP1-his3-621 as the substrate. SIR2 disruption resulted in a smaller-fold increase in meiotic ectopic recombination frequency at RDN1 in the strains carrying TRP1-his3-621 (5- to 8-fold) compared with Ty1mhis3-AI (15- to 30-fold). These differences were probably due to the higher His⁺ recombination levels in the SIR2 strains carrying TRP1-his3-621 at RDN1 and could reflect dependence on the expression of the TRP1 gene.

We propose that all recombination events involving RDN1 (except for intersister exchange) are repressed in a SIR2-dependent manner. However, SIR2 disruption only partially relieved the block of RDN1 to interchromosomal ectopic recombination. One possible cause of sequestration is the physical localization of RDN1 in the nucleolus. Therefore, additional barriers that are unique to interchromosomal ectopic recombination might exist.

What might be the evolutionary significance of recombinational sequestration in meiosis? In mating-type switching, donor choice is nonrandom to ensure productive switching and mating following sporulation. In meiosis, one possibility is that natural direct repeat sequences are inhibited from all ectopic recombination to maintain correct copy number and avoid chromosome translocations. However, CUP1::his3 underwent high levels of interchromosomal meiotic ectopic recombination (Table 3), demonstrating that recombinational sequestration is not a general property of naturally occurring direct repeats. CUP1 might not be subjected to sequestration because it is much shorter than RDN1 and is not part of a specialized intranuclear structure such as the nucleolus.

Alternatively, recombinational sequestration might protect some genomic regions from the consequences of invasion by transposable elements. This model has also been proposed by SMITH and BOEKE 1997 . RDN1 is an efficient target of induced Ty1 transposition (BRYK et al. 1997 ). This phenomenon could result from the large number of repeat units in the array or the tendency of RNA polymerase III-transcribed genes to serve as efficient recipients of Ty1 transposition (KIM et al. 1998 ). If a Ty1 element transposed to RDN1, meiotic ectopic recombination with one of the other ~30 genomic Ty1 elements (CAMERON et al. 1979 ) could disrupt the structure of the nucleolus, resulting in decreased fitness.

Regions other than the rDNA of the S. cerevisiae genome are also sequestered from meiotic ectopic recombination. E. J. LOUIS (personal communication) has observed that telomeres are poor participants in meiotic ectopic recombination with nontelomeric loci. Unlike RDN1, telomeres underwent efficient allelic and interchromosomal ectopic recombination with other telomeres. We surveyed the genome for additional sequestered sites by making insertions of the TRP1-his3-621 cassette (Fig 2B) using restriction endonuclease-mediated illegitimate recombination (SCHIESTL and PETES 1991 ). With that approach we again identified RDN1 and found an additional site on chromosome IV, between Ty1 and Ty2 elements at position D987072, as positions sequestered from meiotic ectopic recombination (E. S. DAVIS and J. N. STRATHERN, unpublished data). Additional investigation is required to determine how many other such sites exist and what functions are required for their sequestration.

	ACKNOWLEDGMENTS

We thank David Garfinkel, Susan Holbeck, Amar Klar, Dwight Nissley, and Alison Rattray for comments on the manuscript. We thank Joan Curcio and David Garfinkel for yeast strains and Mary Bryk and Michael Lichten for plasmids. We also thank Joan Hopkins for administrative assistance. Research was sponsored in part by the National Cancer Institute, Department of Health and Human Services, and the ABL-Basic Research Program. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement from the United States government.

Manuscript received July 21, 1999; Accepted for publication March 20, 2000.

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