Tying Synaptonemal Complex Initiation to the Formation and Programmed Repair of DNA Double-Strand Breaks

Tying synaptonemal complex initiation to the formation and programmed repair of DNA double-strand breaks

Kiersten A. Henderson and Scott Keeney*

Molecular Biology Program, Memorial Sloan–Kettering Cancer Center and Weill Graduate School of Medical Sciences, Cornell University, New York, NY 10021

Communicated by Nancy Kleckner, Harvard University, Cambridge, MA, February 5, 2004 (received for review September 25, 2003) During meiosis, homologous chromosomes recombine and become mutants defective for recombination initiation (21, 22), and in closely apposed along their lengths within the synaptonemal yet other organisms (e.g., females of the silk moth Bombyx mori), complex (SC). In part because Spo11 is required both to make the SC formation in the absence of recombination is the norm (23). double-strand breaks (DSBs) that initiate recombination and to How is the linkage achieved when recombination and SC promote normal SC formation in many organisms, it is clear that formation are tied together? At least a part of this coupling may these two processes are intimately coupled. The molecular nature come through nucleation of SC formation at sites where meiotic of this linkage is not well understood, but it has been proposed recombination is occurring and in particular at the subset of sites that SC formation initiates locally at the sites of ongoing recom- that will give rise to crossovers. There is a one-to-one corre- bination and in particular at the subset of sites that will eventually spondence between synapsis and crossing over for certain un- give rise to crossovers. To test this hypothesis, we examined usual chromosome configurations in maize (24–26) and other further the relationship between DSBs and SC formation in Sac- organisms (27, 28). Based on such findings, Maguire (24–26) charomyces cerevisiae. SCs were monitored in a series of spo11 proposed a causal link between crossing over and synapsis, missense mutants with varying DSB frequencies. Alleles that although the correlative nature of the relationship could not blocked DSB formation gave SC phenotypes indistinguishable from establish which was cause and which was effect. In the fungus a deletion mutant, and partial loss-of-function mutations with Sordaria macrospora, SC initiation is spatially and functionally progressively more severe DSB defects caused corresponding de- coordinated with crossover-designated sites: during zygotene, fects in SC formation. These results strongly correlate SC formation short segments of SC surround crossover-related recombination with Spo11 catalytic activity per se. Numbers of Zip3 complexes on nodules (although not all synaptic initiation sites have an asso- chromosomes, thought to represent the sites of SC initiation, also ciated nodule), and mutants with fewer crossovers have fewer declined when Spo11 activity decreased, but in a markedly non- synaptic initiation events (29). Spatial correlation of crossover- linear fashion: hypomorphic spo11 alleles caused larger defects in related nodules and SC segments was also reported for Neuro- DSB formation than in Zip3 complex formation. This nonlinear spora (30). Finally, in budding yeast, proteins involved in initi- response of Zip3 closely paralleled the response of crossover ating SC formation play roles in generating crossovers. SC recombination products. The quantitative relationship between assembly requires Zip2 and Zip3, which form discrete Spo11- Zip3 foci, SC formation, and crossing over strongly implicates dependent complexes that colocalize with the earliest central crossover-designated recombination intermediates as the sites of element (Zip1) structures and which are thus thought to nucleate SC initiation. central element polymerization (31, 32). Zip2͞Zip3 complexes localize to a subset of recombination sites (31, 32), and the Zip wo prominent features of meiotic prophase are homologous proteins are required for processing crossover-designated re- Trecombination and formation of the synaptonemal complex combination intermediates (33, 51). (SC). Recombination establishes physical connections between Taken together, these studies draw strong connections be- homologous chromosomes in the form of crossovers, which help tween SC initiation and crossover formation. However, several orient chromosomes properly on the meiosis I spindle. The issues arise. The correlative nature of many of these results does widely conserved SC connects homologous chromosomes along not prove a cause-and-effect relationship. Moreover, most of the their lengths during a portion of meiotic prophase and consists studies relied for technical reasons on mutants in which the of two lateral elements, each formed along the axis connecting chromosome structure is grossly perturbed, through either chro- a pair of sister chromatids, and a central element linking the mosome rearrangements or removal of one or more structural lateral elements together (reviewed in refs. 1 and 2). protein components, raising the possibility that the observed Chromosome synapsis is tightly coupled to homologous re- correlations were indirect. Finally, some of the SC–recombina- combination in many organisms. The DNA double-strand breaks tion connection rests on the SC defect in spo11 nulls, but such (DSBs) that initiate recombination occur before synapsis begins studies cannot distinguish whether the synaptic defect stems in budding yeast (3), and cytological features (e.g., histone from the absence of Spo11 catalytic activity (i.e., the inability to H2AX phosphorylation and formation of strand exchange pro- make DSBs) or from the absence of a DSB-independent Spo11 tein complexes) indicate that the same is true in other fungi, function. Recent studies indicate that Spo11 does indeed play GENETICS higher plants, and mammals (4–8). DSBs are created by the roles in meiosis independent of its ability to form DSBs. Spe- Spo11 protein (9, 10), and spo11 null mutants in many organisms cifically, mutation of a tyrosine residue (Tyr-135) responsible for are defective for SC formation (11–16). In some cases, exoge- cleaving DNA abolishes DSB formation (9) but does not affect nous DSBs can at least partially substitute for Spo11 in SC the ability of Spo11 to support normal S phase kinetics or formation (15–17). Moreover, mutations that block processing of homologous chromosome pairing (ref. 34, but see also ref. 35). DSBs once they are formed (e.g., rad50S, dmc1) also cause defects in SC formation (18–20). These results place Spo11 action upstream of SC formation, both temporally and function- Abbreviations: DSB, DNA double-strand break; SC, synaptonemal complex. ally. This coupling is not universal, however; in Drosophila *To whom correspondence should be addressed. E-mail: [email protected]. melanogaster and Caenorhabditis elegans, normal SC is formed in © 2004 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400843101 PNAS ͉ March 30, 2004 ͉ vol. 101 ͉ no. 13 ͉ 4519–4524 Downloaded by guest on September 24, 2021 Moreover, Spo11 is required to recruit Rec102 and Rec104 to meiotic chromosomes independent of DSB formation (52). We addressed these issues by examining SC and Zip3 complex formation in a series of yeast strains that differed with respect to DSB frequency but were otherwise wild type; i.e., they expressed all of the recombination and chromosome structure proteins. To do this, we tested a series of spo11 alleles that reduce or abolish DSB formation without altering steady-state protein levels. Our results reveal that Spo11 promotes SC formation by virtue of its catalytic activity per se, arguing strongly that recombination directly promotes SC formation. Furthermore, the quantitative relationship between SC formation and crossing over in this study strongly supports the model that SC formation initiates locally at the subset of recombination intermediates that are destined to give rise to crossovers. Materials and Methods Meiotic cultures were prepared as described (3, 18). Yeast strains are derivatives of SK1 and are listed in Table 2, which is published as supporting information on the PNAS web site. Tagged spo11-HA3His6 and the spo11 missense mutants were described (36, 37). For simplicity, the tag will hereafter be referred to as ‘‘HA.’’ The spo11-HA allele complements a null spo11 mutation at 30°C, although it confers a mild cold- sensitive phenotype that is recessive to wild type (36). ZIP3-GFP::URA was provided by G. S. Roeder, Yale University, New Haven, CT (32). Details of the subcellular fractionation assay will be provided Fig. 1. (A) Zip1 staining patterns observed in wild-type and͞or spo11 elsewhere (52). Western blotting, and measurements of DSBs mutants. Meiotic chromosome spreads were analyzed by indirect immuno- and intragenic recombination frequencies were performed as ﬂuorescence with anti-Zip1 antibodies. Examples are shown for (i) partial SC, described (36, 37). For SC analysis, chromosomes were surface (ii) extensive SC, (iii) polycomplex, (iv) partial synapsis plus polycomplex, and (v) extensive synapsis plus polycomplex. (Bar, 1 ␮m.) (B) Kinetics of SC forma- spread and stained as described (38, 39). Affinity-purified rabbit tion in spo11-HA and spo11⌬ strains. At least 100 nuclei were scored per time anti-Zip1 antibody (generously provided by G. S. Roeder) was point. used at 1:100 dilution and goat anti-rabbit-Alexa 488 at 1:1,000 (Molecular Probes). For Zip3-GFP analysis, spreads were prepared according to ref. 11, except that cells were treated with anine for the catalytic residue Tyr-135 (9). Two other mutants zymolyase for 10 min at 30°C, and the spheroplast pellet was came from a structure͞function analysis of yeast Spo11 (37). resuspended in a 2:1:1 mixture of 4% paraformaldehyde, 1% Mutation of Asp-288 (thought to directly coordinate a magne- Lipsol, and MEM. Zip3-GFP was detected by using affinity- sium ion) to asparagine compromised DSB formation as severely purified goat anti-GFP at 1:200 (Rockland, Gilbertsville, PA) as a deletion mutant. Substituting alanine for Asp-290 yielded a and donkey anti-goat-Alexa 488 at 1:1,000 (Molecular Probes). partially DSB-defective mutant. These mutant proteins accumu- Zip1 was detected by using affinity-purified rabbit anti-Zip1 at lated to similar steady-state levels as wild type (37), and they 1:100 and donkey anti-rabbit-rhodamine at 1:200 (Molecular behaved similarly to wild type in the differential extraction assay Probes). Electron microscopy was performed as described (11) (Fig. 6C), indicating that they are also chromatin-associated. by using a JEOL 1200EX microscope. Results spo11 Missense Mutants That Do Not Form DSBs Do Not Form SC. To determine SC phenotypes, meiotic chromosome spreads were Wild-Type and Mutant Spo11 Proteins Are Associated with Chromatin. immunostained for Zip1, a component of the SC central element Given its role in promoting DSB formation, Spo11 should be (40). Spread nuclei were assigned to classes according to the associated with chromatin. To confirm this and to determine Zip1 pattern: no staining or faint diffuse Zip1 staining; bright whether DSB-defective mutant Spo11 proteins are still capable Zip1 foci; partial SC (at least one linear stretch of Zip1 staining; of localizing to chromatin, we used a differential extraction assay ͞ to follow Spo11 subcellular distribution (diagrammed in Fig. 6, i.e., zygotene); or extensive SC (late zygotene pachytene). Ex- which is published as supporting information on the PNAS web amples of nuclei with partial or extensive SC are shown in Fig. site). Meiotic cells were spheroplasted, hypotonically lysed, and 1Aiand ii. A typical time course in a spo11-HA strain is shown then separated by centrifugation into a soluble cytoplasmic in Fig. 1B. The kinetics and frequency of SC formation in this fraction and a pellet containing nuclei and other insoluble strain were indistinguishable from an isogenic wild-type strain material. The pellet was then sequentially extracted with non- (data not shown; see also below) and are consistent with earlier ionic detergent and with DNase I digestion. We define a protein studies in this strain background (3). as being chromatin-associated if it is enriched in the pellet Consistent with earlier electron microscopic studies (12, 13), ⌬ containing the chromosomes (P2) and then solubilized by DNase a spo11 mutant showed severe SC defects. Nuclei with partial I treatment (S3). Tubulin is in the first soluble extract (S1) and or extensive SC formation were entirely absent (Fig. 1B). serves as a control for the efficiency of spheroplast formation Somewhat surprisingly, nuclei containing bright Zip1 foci ap- and lysis. At4hinmeiosis, this analysis revealed that a significant peared at near-normal frequency and with normal kinetics, fraction of cellular Spo11 protein is associated with meiotic indicating that a significant fraction of such foci probably do not chromatin (Fig. 6B). represent sites of synaptic initiation and may instead be Zip1 We chose several spo11 mutants for the work presented here. aggregates bound nonspecifically to chromatin. Two missense In one, DSB formation was abrogated by substituting phenylal- mutants completely defective for DSB formation, spo11-

4520 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400843101 Henderson and Keeney Downloaded by guest on September 24, 2021 Fig. 2. Synaptic defects in spo11 mutants with reduced DSB levels. Chromosome spreads from the indicated strains were immunostained for Zip1. At least 50 nuclei were scored at each time point. Approximate relative DSB activities (from Table 1) are provided for convenience. (A) Extent of SC formation. (B) Occurrence of nuclei containing polycomplexes (PC), with or without partial or extensive synaptonemal complex.

Y135F-HA and spo11-D288N-HA, also showed severe SC defects formation, although 5–10% of cells eventually made at least (Fig. 2Avand vi). Similar results for spo11-Y135F were reported short SC stretches (Fig. 2Aiii); and the configuration with very earlier (B. Weiner and N. Kleckner, unpublished results; see ref. low recombination (spo11-Y135F͞spo11-HA) showed a profound 34). These findings support the idea that SC formation is closely SC defect that was only marginally better than completely tied to DSB formation per se. DSB-defective mutants (Fig. 2Aiv). Thus, partial DSB defects cause partial synaptic defects but only after a threshold in DSB spo11 Mutants with Partial DSB Defects Show Partial Synaptic De- levels is crossed. fects. We next examined an isogenic set of strains with progressively more severe DSB defects. One strain was homozygous for Polycomplex Formation. Polycomplexes are aggregates of SC com- spo11-D290A-HA, which reduces DSB activity in vivo to Ϸ4– ponents that form at increased frequency in strains unable to 12% of wild type, as estimated from direct DSB measurements form proper SC (13, 18, 19, 31, 41–43). Because they can be and from frequencies of intragenic recombination at the easily visualized by Zip1 immunofluorescence (Fig. 1A iii–v), his4::LEU2 hot spot (Fig. 3 and Table 1). Two other strains took advantage of dominant negative effects of spo11-Y135F (37). The extent of the DSB defect in a spo11-Y135F͞ϩ heterozygote depends on the precise combination of epitope tags on the two alleles. If both alleles are tagged, the Y135F mutation is semidominant and the strain shows 17–23% of wild-type recombination initiation (Fig. 3 and Table 1). However, if the wild-type allele is tagged and the point mutant untagged (spo11-Y135F͞ spo11-HA), the mutation is almost completely dominant and the strain shows Ͻ5% of normal recombination initiation (Fig. 3 and Table 1). The reason for these effects is not known, but for purposes of this study, we can view these allelic combinations simply as a way to titrate Spo11 activity in vivo. Pulse-field gel analysis revealed that these mutations affect DSB frequencies to GENETICS similar extents across several regions of the genome (R. Diaz and S.K., unpublished observations), so it is likely that measurements at a single recombination hot spot provide a reasonable estimate of relative DSB levels in the genome as a whole. The reduction in DSB frequencies in a spo11-HA strain (to Ϸ 30–60% of wild type) caused no detectable defect in SC Fig. 3. DSBs in a SPO11 allelic series. Genomic DNA was prepared at the formation, but further reductions in DSB levels correlated with indicated times from rad50S strains differing in their genotype at the SPO11 reduced SC formation. The semidominant negative mutant locus. DSBs at the his4::LEU2 locus were analyzed by Southern blotting of (spo11-Y135F-HA͞spo11-HA) formed SC at a reduced frequency PstI-digested DNA. Note that the spo11-D290A mutation affects both the level (Fig. 2Aii); spo11-D290A-HA gave no cells with extensive SC and distribution of DSBs within the hot spot, as described (37).

Henderson and Keeney PNAS ͉ March 30, 2004 ͉ vol. 101 ͉ no. 13 ͉ 4521 Downloaded by guest on September 24, 2021 Table 1. Relative recombination frequencies for various spo11 mutants Frequency Hisϩ, ϫ103 rad50S DSBs, percent of total DNA SPO11 genotype (percent of wild type)* (percent of wild type)†

SPO11͞SPO11 18.9 Ϯ 3.6 (ϵ 100)‡ 9.8 Ϯ 1.8 (ϵ 100) spo11-HA͞spo11-HA 11.8 Ϯ 5.8 (62)‡ 2.6 Ϯ 0.40 (27) spo11-Y135F-HA͞spo11-HA 4.4 Ϯ 2.2 (23)‡ 1.7 Ϯ 0.34 (17) spo11-D290A-HA͞spo11-D290A-HA 0.84 Ϯ 0.35 (4.4) 1.2 Ϯ 0.27 (12) spo11-Y135F͞spo11-HA 0.12 Ϯ 0.046 (0.63)‡ 0.42 Ϯ 0.08 (4.3)

*Intragenic recombination at his4X::LEU2͞his4B::LEU2, measured as the frequency of Hisϩ prototrophs per viable cell after8hinsporulation medium (mean Ϯ SD of at least three determinations). †DSB frequencies were measured at his4LEU2 site I in rad50S derivatives of the strains used for return-to-growth measurements (mean Ϯ SD of at least two determinations). ‡Data are from ref. 37.

polycomplexes are useful indirect indicators of synaptic defects. the normal pattern of SC formation noted above for this strain. Whereas polycomplexes were rare in wild-type or spo11-HA cells As Spo11 activity was decreased further, however, the number of (Fig. 2Bi and data not shown), polycomplexes were prevalent in Zip3 complexes declined. In spo11-Y135F-HA͞spo11-HA, signif- spo11⌬ cells (data not shown), in agreement with earlier studies icantly fewer Zip3 foci relative to wild type were observed in (13). Similarly, DSB-null missense mutants (spo11-Y135F-HA nuclei containing partial (zygotene) or full (pachytene) SCs (Fig. and spo11-D288N-HA) frequently yielded nuclei that contained 5B, P Ͻ 0.001, Wilcoxon rank sum test). In the more severely only polycomplex with no other obvious Zip1 structures, and the compromised strain (the spo11-D290A-HA homozygote), Zip3 dominant-negative configuration (spo11-Y135F͞spo11-HA) was focus counts were reduced even further relative to wild type (P ϭ again essentially indistinguishable from these (Fig. 2Biv–vi). 0.006 for the nuclei with only focal Zip1 staining patterns; P Ͻ Strains with intermediate DSB frequencies (spo11-Y135F-HA͞ 0.001 for nuclei with partial SC). Zip3 foci were almost entirely spo11-HA or spo11-D290A-HA) showed intermediate polycom- absent in a spo11-Y135F-HA mutant, as for spo11⌬ (data not plex formation that progressively worsened as the DSB defect shown). These results agree well with the SC phenotypes of these worsened. Interestingly, these strains frequently showed poly- mutants, so it is likely that the synaptic defects can be directly complex simultaneously with SC (Figs. 1Aivand v and 2Biiand attributed to decreases in the number of synaptic initiation sites. iii). Such nuclei were rare in wild type (Fig. 2Bi). The inverse It is important to note, however, that the relationship between correlation between DSB frequency and polycomplex formation DSB frequencies and the number of Zip3 complexes is distinctly further reinforces the connection between Spo11 catalytic ac- nonlinear (Fig. 5C). Implications of this observation are dis- tivity and chromosome synapsis. cussed below.

Confirmation of Aberrant Synaptic Structures by Electron Microscopy. Discussion To confirm the structures classified by Zip1 immunofluores- To test whether recombination promotes SC formation, we used cence, chromosome spreads were also analyzed by silver staining a panel of yeast spo11 missense mutants that show a range of and electron microscopy. In agreement with Zip1 immunoflu- DSB frequencies. The mutant proteins localized properly to orescence, cells with a partial Spo11 defect often showed SC simultaneously with polycomplexes (Fig. 4 B and C). Of Ͼ100 spo11⌬ nuclei examined, none showed SC, but many contained duplicated unseparated spindle pole bodies and polycomplex (Fig. 4D). None of the spo11⌬ nuclei contained obvious structures resembling axial elements. Similar observations were made for the completely DSB-defective mutant spo11-Y135F-HA (data not shown).

Reducing DSB Formation Decreases the Number of Zip3 Complexes. As described in the introduction, Zip3 complexes are thought to mark the sites of SC initiation (31, 32). If the main contribution of Spo11 to SC formation is to provide these initiation sites, then the number of Zip3 complexes on chromosomes should decrease coordinately with decreasing Spo11 activity. Chromosome spreads were prepared from four isogenic strains varying in their SPO11 genotype, all expressing a Zip3-GFP fusion (32). The spreads were stained with anti-Zip1 and anti-GFP antibodies, then at least 50 nuclei were selected at random from each of five time points (2–6 h in meiosis), grouped according to the extent of SC formation, and the numbers of Zip3 foci were counted. Representative nuclei are shown in Fig. 5A, and the numbers of Zip3 foci are shown in Fig. 5B. In agreement with previous studies (32), Zip3 foci first Fig. 4. Electron microscopic conﬁrmation of normal and aberrant synaptic structures. Chromosome spreads prepared at4hinmeiosis were stained with appeared in wild type at or before the time when the first Zip1 silver and analyzed by electron microscopy. (A) Extensive SC in a spo11-HA staining structures appeared, and they increased in number as nucleus. (B and C) Extensive SC plus polycomplex (B) and partial SC plus SC formation progressed (Fig. 5B, wild type). No significant polycomplex (C), both from a spo11-Y135F-HA͞spo11-HA strain. (D) Polycom- differences in the number or timing of Zip3 complex formation plex only, from a spo11⌬ nucleus. Arrows, spindle pole bodies; arrowheads, were observed in spo11-HA compared to SPO11, consistent with polycomplexes. (Bar, 0.5 ␮m.)

4522 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400843101 Henderson and Keeney Downloaded by guest on September 24, 2021 Fig. 5. Decreased numbers of Zip3 foci form when DSB formation is reduced. (A) Representative nuclear spreads stained for Zip1 (red) and Zip3-GFP foci (green). (Bar, 1 ␮m.) (B) Zip3 focus counts. For each strain, nuclear spreads were prepared hourly from 2–6 h in meiosis. At least 50 nuclei were selected at random for each time point and grouped according to the extent of SC formation irrespective of the presence of polycomplex, and the Zip3 foci were counted. Bar graphs show the percent of nuclei that contained the indicated number of Zip3 foci (0–9 foci͞nucleus in pale yellow, etc.). The number of nuclei in each Zip1 class (n) and the mean number of Zip3 foci (ϮSD) are given (Right). **, For spo11-D290A-HA, only one nucleus (with 28 Zip3 foci) had extensive SCs. (C) Nonlinear relationship between DSB frequency and the number of SC initiation sites. Relative numbers of Zip3 foci are plotted against relative DSB frequencies for wild-type, spo11-HA, spo11-Y135F-HA͞spo11-HA, and spo11-D290A-HA (⅙). Zip3 numbers are the averages for all nuclei scored as having either partial or extensive SC (from B). The gray line marks the diagonal expected for a linear relationship.

meiotic chromosomes and supported at least some of the normal scribed: increases in crossing over caused by loss of Sgs1 function functions carried out by wild-type Spo11. Two different point in yeast were accompanied by increases in numbers of synaptic mutations that abrogated DSB formation blocked SC formation initiation sites (Zip3 complexes) (44). The idea that SC forma- as severely as a deletion mutation. Our results agree with other tion initiates primarily, if not exclusively, at the subset of studies of the spo11-Y135F mutant in yeast (N. Kleckner and B. recombination events that are destined to become crossovers Weiner, personal communication and unpublished data; see ref. provides a simple way to explain the similar responses of Zip3 34) and an equivalent mutant in S. macrospora (8). We also found foci and crossover numbers in these two studies. Moreover, this that mutants with intermediate DSB defects gave intermediate explanation is consistent with the implication of Zip2 and Zip3 SC defects. Because several different missense mutations and in controlling the processing of crossover-designated recombi- allele combinations were used, it seems unlikely that we man- nation intermediates (51) and with the observation that the aged fortuitously to covary two independent functions of Spo11. number of Zip2͞Zip3 foci per cell is sufficient to account for Thus, these findings strongly link the DNA-cleaving activity of only a subset of the total DSBs (31, 32). Spo11 to its activity in promoting SC formation. Our observations agree well with recent studies of the effects of hypomorphic ski8 alleles on SC formation in S. macrospora Crossover-Designated Recombination Sites Initiate Synapsis. The (17). These findings, along with earlier studies in S. macrospora, results presented here indicate that the major role for Spo11 in N. crassa, and maize (see Introduction), make it likely that SC formation is to provide the sites where Zip3 loads onto preferred nucleation of central element formation at crossover- chromosomes and nucleates Zip1 polymerization. More impor- designated intermediates is a general phenomenon in many tantly, the results also support the idea that synapsis initiates at organisms. It is also clear that this is not the only mechanism, a defined subset of recombination intermediates with properties however. For example, plants with very long chromosomes have characteristic of crossovers. This conclusion is based on the more SC initiation sites than crossovers (45, 46); SC initiation finding that the effects of spo11 mutants on Zip3 focus numbers sites without associated recombination nodules are observed correlate better with effects on crossover frequencies than with near telomeres in S. macrospora (29); SC formation can occur in effects on total DSB levels. In separate studies, crossover spo11 mutants in mice, albeit between nonhomologous chromo- frequencies were analyzed in a similar SPO11 allelic series as was somes (14, 15); and SC formation occurs normally in the absence used here. Surprisingly, even as Spo11 activity decreased, cross- of recombination in a number of organisms (see Introduction). over frequencies did not decrease in parallel (E. Martini, R. Diaz, N. Hunter, and S.K., unpublished observations). It thus How Defective for SC Formation Are spo11 Mutants in Yeast? In some appears that cells have a mechanism to maintain appropriate studies of spo11 and other DSB null mutants in budding yeast, numbers of crossovers despite variation in the frequency of no SC was detected (12, 18, 43, 47, 48). In others, at least limited GENETICS recombination initiation. We refer to this phenomenon as SC formation could be detected, although it is not clear whether ‘‘crossover homeostasis.’’ this was homologous or nonhomologous synapsis (refs. 13 and 49 As the DSB frequency decreased, the number of Zip3 foci also and K. Schmekel, personal communication). Despite the differ- decreased, but the two did not vary together in a linear fashion. ences, it seems fair to say that no study has led to the conclusion Specifically, Zip3 foci showed a threshold effect (Fig. 5C), that SC formation is normal in these mutants, and almost all indicating that the number of Zip3 foci is buffered against studies document profound defects. We suggest that the pre- changes in DSB frequency. This behavior is quantitatively ferred pathway for SC formation in yeast is to install the central indistinguishable from crossover homeostasis. While this paper element first at the sites where crossovers will occur, but that was in preparation, a striking converse correlation was de- limited Zip1 assembly can occur elsewhere under at least some

Henderson and Keeney PNAS ͉ March 30, 2004 ͉ vol. 101 ͉ no. 13 ͉ 4523 Downloaded by guest on September 24, 2021 conditions (variations in culture conditions, strain background, 50). Although a single such break could not induce substantial temperature, etc.). Given the rarity of recombination- SC formation (50), these studies most likely would not have independent synapsis under most conditions, it seems likely that detected formation of a short stretch of SC, and formation of this pathway contributes relatively to SC formation in normal Zip2͞Zip3 foci was not analyzed (J. Haber, personal commu- meiosis. nication). Thus, it will be interesting to determine whether a The ideas presented here predict that SC should form at any single DSB can promote local SC initiation. DSB if it is being processed along an appropriately controlled crossover-designated pathway. DSBs induced by exogenous We thank Doug Bishop (University of Chicago, Chicago), Mike Dresser DNA damage can rescue SC formation in spo11 mutants in (Oklahoma University, Oklahoma City, OK), and Shirleen Roeder (Yale University, New Haven, CT) for providing strains, antibodies, and͞or several organisms, so recombination need not be initiated by technical advice; Nancy Kleckner for critical comments on the manu- Spo11 to promote SC formation (15–17). An endonuclease- script; Adam Olshen for help with statistical analysis; and Nina Lampen mediated DSB can be repaired to generate a crossover in yeast, for help with electron microscopy. This research was supported in part with many of the hallmarks of Spo11-induced recombination (35, by National Institutes of Health Grant GM58673.

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