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One possibility is that high levels of cohesin turn the sister chromatid within pericentromeres into a preferred repair template. Indeed, it has been shown that template choice sister or homologue for DSB repair during budding yeast vegetative growth is dictated by the levels of cohesin Covo et al. Globally, on meiotic chromosomes, repair of DSBs is normally biased towards use of the homologous chromosome instead of the sister chromatid to ensure at least one COs is formed to link each homolog pair Hollingsworth, ; Humphryes and Hochwagen, This signaling pathway is thought to locally antagonize cohesin to enable the use of the more distant homologous chromosome as a repair template Hong et al.

Our findings point to a second mechanism through which cohesin steers repair of meiotic DSBs away from inter-homologue repair: by promoting the local recruitment of Zip1. Zip1 has previously been implicated in preventing pericentromeric CO and NCO repair, and was suggested to promote inter-sister repair Chen et al. Zip1 performs specific functions at centromeres during prophase, which include non-homologous centromere-coupling Tsubouchi and Roeder, ; Tsubouchi et al.

Here we have shown that the Ctf19 complex, presumably through its role in directing pericentromeric cohesin enrichment, enables the dedicated recruitment of a centromere-localized pool of Zip1 to perform these specialized functions. Overall, we conclude that the kinetochore, and specifically the Ctf19 complex, promotes the establishment of an inter-homologue recombination-suppressed zone surrounding centromeres.

To do so, the Ctf19 complex recruits high levels of cohesin within pericentromeres, which in turn triggers efficient recruitment of the synaptonemal complex component Zip1, effectively leading to strong, local inhibition of inter-homologue directed repair.

Mitotic crossing over and nondisjunction in translocation heterozygotes of Aspergillus.

Mechanistically, pericentromeric CO formation has been suggested to lead to a local disturbance of sister chromatid cohesion, which could lead to precocious separation sister chromatid, causing meiosis II non-disjunction and aneuploidy Rockmill et al. While meiosis II mis-segregation is a characteristic feature of Ctf19 complex mutants Fernius and Marston, , the impact of pericentromeric COs on this phenotype is currently unclear because of the requirement for this complex for proper pericentromeric cohesion.

The kinetochore is a sophisticated machine that couples chromosomes to microtubules and drives their segregation in meiosis and mitosis. The kinetochore also serves as a signaling platform that monitors and responds to the state of kinetochore—microtubule attachment in the context of the cell cycle. During meiosis, in addition to the canonical events that also take place during mitosis, the kinetochore takes on additional roles to bring about the specialized segregation pattern.

First, sister chromatids need to attach to microtubules that emanate from the same pole in a mono-oriented fashion. Second, sister chromatid cohesion at pericentromeres needs to be protected from removal during meiosis I. In both cases, the kinetochore controls these events by coordinating the recruitment of specific protein complexes i. Our data add a hitherto unknown, additional level of functionality to the kinetochore in meiosis, in which it impacts meiosis-specific CO formation by influencing both meiotic DSB formation and recombinational break repair. As such, it effectively prevents the formation of unwanted chiasma within pericentromeres.

Finally, we note that our data might also provide an additional rationale for why much higher cohesin levels need to be established around centromeres, namely to minimise CO formation near centromeres. The Ctf19 complex is a generally conserved component of eukaryotic kinetochores. Suppression of meiotic CO recombination within pericentromeres is a widespread feature of meiotic recombination in many diverse organisms, whether or not their centromeres are surrounded by large blocks of heterochromatinized DNA.

We therefore suggest that Ctf19 complex-driven suppression of meiotic CO formation serves as a universal component of the mechanisms that shape the meiotic recombination landscape in order to promote the faithful propagation of the genome from generation to the next. Yeast strains used in this study are derivatives of SK1 and genotypes are given in Supplementary file 4A except for the strains used for analysis of recombination genome-wide where AM and AM haploid strains derived from YJM and S96, respectively, were used.

Standard techniques were used to generate gene deletions, promoter replacements and epitope-tagged proteins. Ohta Kugou et al. For the anchor away system, parental SK1 strains were generated as described by Haruki et al. Cells were washed once with sterile distilled water and re-suspended in SPO media 0. Cells were incubated at 30 o C for the duration of the experiment.

Prophase I block-release experiments were performed as described by Carlile and Amon Western blotting was performed as previously described Clift et al. Using BWA Version: 0. Duplicate reads were removed for parallel analysis using SAMtools Version 1. The data shown were normalized to the number of reads per million of total mapped reads.

The total mapped reads were established after any processing. Chromosome spreading was performed as described previously Loidl et al. The camera, shutters, and stage were operated through SoftWorx software Applied Precision. Samples for studying GFP-labelled chromosomes were prepared as described previously Klein et al. Yeast strains were placed on SPO agar plates 0. After 4 days, images were captured in three channels and the pattern of fluorescence scored in the tetrads.

To prevent confounding effects due to chromosome mis-segregation a common occurance in kinetochore mutants , only tetrads where all 4 spores had acquired CFP blue fluorescence were included in the final analysis. All raw data and statistical analysis is given in Supplementary files 1 and 2. Southern blotting was performed as previously described Vader et al. CEN1:I , ,—, DSB intensities were analysed using ImageJ. Flow cytometry was performed as described Vader et al. Identification of single nucleotide polymorphisms by high-throughput sequencing was carried out as described by Oke et al.

An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent see review process. Similarly, the author response typically shows only responses to the major concerns raised by the reviewers. Thank you for submitting your work entitled "The kinetochore prevents centromere-proximal crossover recombination during meiosis" for peer review at eLife.

The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission. Meiotic recombination is suppressed in the vicinity of the centromere in many organisms. Studies in budding yeast have shown that this suppression extends not only to crossovers, but to the double-strand breaks DSBs that initiate meiotic recombination. Further mutant studies may be beyond the scope, but the involvement of other kinetochore proteins should be considered and discussed. For example, is the MIND complex retained at meiotic centromeres in the absence of the Ctf19 complex?

If at all possible, additional mutants should be included or additional analysis done so that there is more overlap between figures. For example, the addition of ctfdel to the ChIP-qPCR in Figure 4D, E would provide a useful point of comparison to the anchor-away allele used in Figure 5 , and thus would provide at least an indirect measure of the efficacy of the anchor away.

Similarly, it would be useful to make a direct comparison between COs and DSBs in the same CEN8-associated interval in the mmsdel mutant background. For tetrad analyses, this means that the primary data should be included in a supplementary table, so that statistical power can be evaluated as well as significance of difference. There is a similar concern with Figure 5G. Given that the spore color assay makes it possible to readily examine hundreds of tetrads, it is important to analyze enough to exclude biologically significant differences that might be masked due to small numbers.

Please be aware that, upon revision, if it still appears that insufficient tetrads have been analyzed, there will likely be a direct request to analyze more. In critical cases, in particular the comparison between anchor-away at 0 and 3 hr, DSBs must be quantified. A The extent of Ctf19 depletion at kinetochores is not assessed-this is an important control to do. B It appears that Ctf19 anchor-away does not result in a loss of pericentric cohesin regardless of when it is executed. Similarly, it appears that there are significant losses of cohesin at CEN4 regardless of the time of execution.

Therefore, if there really are differences in DSB repair under the two anchor-away conditions see point 4, above , it is difficult to see how these can be attributed to differences in cohesin recruitment. C To recapitulate point 4, it is not at all clear that crossovers and DSBs are differently affected by the time when Ctf19 anchor-away is executed. Careful quantification is necessary to prove this point, and if the DSB analysis was only done once, additional replicates will need to be done. In addition to the "fold-change" graph in Figure 3B , it would be useful to plot the mean Spooligo signal as a function of distance from the centromere, comparing wild-type and mcmdel.

See Figure 4C in Buhler et al. Are these data compromised by insufficient numbers? As with tetrad data, the actual number of events need to be reported, so that they can be vigorously analyzed for sufficient statistical power and statistical significance. A Ctf19 complex mutants seem to disrupt not only initial Zip1 recruitment to centromeres, but subsequent synapsis.

It's not clear why this should be the case, if arm recombination and cohesin loading are unaffected, and this should be addressed. B While zip1-del mutants increase pericentromeric crossovers and possibly zip1-S75E, if sufficient numbers of tetrads are examined , their impact on pericentromeric DSBs is not addressed, and should be. C The scc4-m35 mutant almost entirely eliminates Zip1 loading at centromeres, but increases pericentromeric crossovers to a substantially greater extent than does zip1-del , suggesting that pericentromeric cohesin might be doing other things to suppress crossovers other than recruiting Zip1.

This should be considered and discussed, and an epistasis experiment should be considered if you truly believe that this is the main mode of pericentric crossover suppression. It would be interesting to examine the role of the MIND complex in CO suppression, however, there are considerable technical difficulties with doing so and this is beyond the scope of the current study. MIND is known to be essential for kinetochore-microtubule attachments and, consistent with this, depletion of its Mtw1 and Dsn1 components during meiosis led to a near-complete failure in chromosome segregation with very few tetranucleate cells being produced this data is now included as Figure 1—figure supplement 1.

Since measurement of CO frequency relies on the production of spores, we were unable to assay CO suppression in these strains. We found that Dsn1 is also required for suppression of DSBs, suggesting the integrity of the kinetochore might be generally important for DSB suppression.

Nondisjunction and chromosomal anomalies

Conversely, Mcm21 was retained at centromeres in Dsn1 cells and appears elevated in Mtw1-depleted cells, despite massive chromosome mis-segregation in these cells Figure 1—figure supplement 1. This suggests a partial and complex inter-dependence of the Ctf19 and MIND complexes at the kinetochore. Although we agree that it will be important to understand the relationship between the Ctf19 complex and MIND in CO suppression in the future, further analysis is beyond the scope of this study.

This analysis shows that there is an increase in DSB levels within the CEN8- associated interval of about 5-fold over wild type. The total increase in CO formation within this interval was fold over wild type Figure 3F and Figure 1B , lending further support for a dual effect of the Ctf19 complex on recombination outcomes at pericentromeric regions see also further below. We thank the reviewers for the helpful suggestions related to the tetrad data and have now done extensive further analyses, which have allowed us to strengthen our conclusions.

As suggested, we have now used the online tool to calculate standard errors and these are given on all tetrad data throughout the manuscript. We have also determined statistical power for all tetrad data: all significant effects reported have a statistical power of at least 0. All raw data is now included in Supplementary file 1 , together with power calculations and statistical analysis.

It is important to note that although in principle it is possible to score hundreds of tetrads in this assay, this is not feasible for many of the strains in practice. Furthermore, chromosome segregation is greatly impaired in Ctf19 complex mutants and to avoid confounding effects of mis-segregation, we can only score tetrads where each spore inherits one copy of the chromosome VIII 4 blue spores , a relatively small fraction of the overall spore count, which is already low. Finally, we found no evidence for significant changes in CO frequency on the chromosomal arm region in Ctf19 complex mutants compared to wild type Figure 1C , though the values appear slightly elevated.

Power calculations indicate that at least tetrads per individual strain containing a faithfully segregated chromosome VIII would need to be analyzed to determine significance for these differences. Due to the difficulties mentioned above, this would be a huge undertaking that would require weeks of analysis for a single strain.

Chromosomal crossover in Meiosis I

We have now repeated and replaced some of the southern blots to improve quality Figures 4B and 6J , and for others we have subtracted general background to increase clarity. These similar levels of DSB formation soon after rapamycin addition but irrespective of the time in meiosis at which Ctf19 is anchored away, is in contrast with the significant differences in CO levels that we observed when anchoring-away Ctf19 using identical regimes Figure 6K. The shown southern analysis and quantification is a representative experiment; a similar experiment was performed that demonstrated comparable effects on DSB intensities and anchoring away of a different Ctf19 complex, Iml3 showed a similar effect on DSB formation.

However, due to technical difficulties in quantifying these blots, in part due to weak specific signals together with high background signals, we have decided to only include the DSB quantification of the southern blot that is shown in Figure 6H. Also some regions of the chromosomes may be so diverged that homology is no longer recognized at the strand exchange stage of recombination see also below.

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To determine if PMS1 and MSH2 are operating in the same or different pathways during homeologous exchanges, a pmsl msh2 double-mutant strain was constructed. Because Pmslp and Msh2p are proposed to act in concert, we would have predicted that a double mutant would be no more severe than either mutant alone. Analysis of post- meiotic segregation frequencies and rates of mitotic mutation support this proposal.

However, with respect to meiotic homeologous recombination, the phenotype of the double mutant is more severe. In fact, the rates of exchange in the double mutant are additive. The correlation between greater crossing over and decreased aneuploidy indicates that many of the crossovers restored in the mutant hybrids can ensure disjunction, that is, can form functional chiasmata a cytological manifestation of crossing over. This relationship between crossover frequency and chromosome disjunction is not linear. From a comparison with S. For example, the medl mutation, an allele of the DMCI gene, has a 2-fold decrease in meiotic crossovers, 4.

It must also be considered that the reduced fidelity of recombination in mismatch repair deficient hybrids may increase the frequency of crossovers between related, ectopic loci. Ectopic crossovers are known to interfere with homologue disjunction and are likely to produce lethal, unbalanced transiocations. One such translocation, giving rise to a unique sized chromosomal species has been observed in a segregant from the msh2 hybrid not shown. The increased spore viability appears to be a direct consequence of improved chromosome disjunction which in turn is the result of increased frequencies of meiotic recombination.

The spore viability of the hybrids are lower than expected from the patterns of disomy observed. The average frequency of disomes in random spores from the msh2 hybrid is 3. The fact that spore viability is not restored to this level indicates that other factors probably contribute to the meiotic sterility of the S. The observation that some S. How the mismatch repair system processes mismatched recombination intermediates at the molecular level is not clear.

Several models have been proposed. The "killer mechanism" causes the destruction of intermediates which could potentially lead to chromosomal loss. Mismatch repair-induced recombination may lead to chromosomal rearrangement or loss. Lastly, antirecombination and the similar "heteroduplex rejection" models propose that intermediates are aborted via disassembly, or resolution without exchange.

From the data presented here none of these possibilities can be excluded. However, the low frequencies of meiotic exchange and high levels of aneuploidy are most consistent with an antirecombination mechanism. Several observations, from a variety of experimental approaches, suggest that recombination intermediates are disrupted at an early stage, prior to the formation of a stable heteroduplex junction.

Firstly, individual components of the bacterial mismatch repair system can block in vitro homologous strand exchange catalyzed by the E. Also recombination intermediates that have been detected during meiotic prophase I, in S. Finally, F1 hybrids between species of Allium that lack gross chromosomal rearrangements have a substantial reduction in the frequency of chiasma and an increased number of univalents at pachytene.

Mutations in the mismatch repair genes MSH2 and MSH3 have been shown to increase the frequency of mitotic homologous recombination, in S. In the data presented here, pmsl produces up to a fold enrichment in meiotic recombinants. The effect of msh2 is significantly greater, not only in terms of recombination, but also for disomy and spore viability. This observation suggests that the method of processing recombination intermediates depends upon the degree of divergence between the participating molecules. From the known biochemical properties of the E.

A number of features of meiotic homologous recombination follow from this assumption. The fact that reasonable frequencies of recombination are observed in pmsl and msh2 hybrids suggests that the induction of meiotic recombination is still high. Also, consistent with the in vitro properties of E. All S. Genotypes are described in Table I. The ho-ochre mutation was isolated by UV- mutagenesis but was found to have a slightly leaky phenotype. Subsequently, heterothallic strains were obtained by creating a bp Pstl deletion of the coding sequence of the HO gene. The A pmsl mutation is a 2.

Both were introduced by one-step gene transplacement. All were introduced by two-step gene replacement. Other auxotrophic markers were spontaneous or UV-induced. All transformations were verified by Southern blot analysis using the digoxigenin, nonradioactive system as recommended by the manufacture Boehringer Mannheim. The resulting strain had this genotype. Yeast manipulations and media were as described in the literature. Germination was scored microscopically after 3 days. Only spores that formed microcolonies were scored as being viable.

One and two-step gene replacement was performed. Yeast transformation was carried out using a modification of the lithium acetate method. Strains disomic for chromosome III, which arise from the chromosome transfer event, were confirmed physically by the appearance of a band of double intensity by CHEF gel analysis and genetically by a nonmating sporulation-deficient phenotype. Transplacement occurs preferentially into the Y55 copy of the MSH3 gene because of reduced homology with the S.

Karyotyping of Segregants Random spore segregants were karyotyped. Disomy was assigned via band intensity or the presence of two bands for co-migrating and polymorphic chromosomes respectively. Data Analysis Data sets were analyzed using the standard normal, nonparametric sign and G-tests. The G-test is an equivalent to the X 2 contingency test.

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Expected distributions of disomes were calculated using the average disomy frequencies in a binomial expansion involving 9 or 16 chromosomes. E 0 values were calculated assuming recombination rates in random spores are equivalent to map distance and a poisson distribution of the number of crossovers per chromosome. Figure 1. A model of the biological consequences of antirecombination during meiosis A. Homologous chromosomes recombine and undergo crossing over.

The homologues become physically connected by a chiasmata and consequently orientate correctly on the meiosis I spindle. Correct disjunction in the first division is followed by an equational division to produce four euploid spores. Spores b. The mismatch repair proteins will prevent a crossover between homologous chromosomes.

Apposition of the centromeres is not ensured and the resultant univalents segregate randomly with respect to each other at meiosis I. If both univalents attach to the same spindle nondisjunction will result. After meiosis n two disomic and two nullosomic spores will be produced. None of the chromosomes will be recombinant.

The nullosomic cells lack essential genetic information and will be dead. The disomic cells contain unbalanced genomes and may have reduced fitness. Y5S S. NHPD2 sjf. NHD50 S. NHD53 S. NHD95 S. NHD47 S. YH2 his4-Rl le. NHD45 Sx. NHD94 Sx. URA3 This study. Abbreviations: S. Strains were constructed as described in Methods and Materials. Diploids were sporulated and tetrad ascospores dissected. To reduce the spore death caused by the mutator phenotypcs of pmsl and msh2, vegetative growth as a diploid was minimised. All strains were treated in this way.

Table III Frequency of disomes in hybrid segregants. Random segregants were karyotyped by CHEF gel electrophoresis. Chromsome I. Randoa spores were analysed for recoabination in the four intervals shown. Map distance is equivalent to the frequency of recombinants. None of the intervals in the control diploids, NHD50, 53 and 95 are statistically different by standard normal test. Special Interest For the Children He sat military to Manage it away from total recombination and meiosis crossing over and disjunction.

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