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Separable Crossover-Promoting and Crossover-Constraining Aspects of Zip1 Activity during Budding Yeast Meiosis.

Voelkel-Meiman K, Johnston C, Thappeta Y, Subramanian VV, Hochwagen A, MacQueen AJ - PLoS Genet. (2015)

Bottom Line: While stable, full-length SC does not assemble in S. cerevisiae cells expressing K. lactis ZIP1, aggregates of K. lactis Zip1 displayed by S. cerevisiae meiotic nuclei are decorated with SC-associated proteins, and K. lactis Zip1 promotes the SUMOylation of the SC central element protein Ecm11, suggesting that K. lactis Zip1 functionally interfaces with components of the S. cerevisiae synapsis machinery.This separation-of-function version of Zip1 thus reveals that neither assembled SC nor MutSγ is required for Mlh3-dependent crossover formation per se in budding yeast.Our data suggest that features of S. cerevisiae Zip1 or of the assembled SC in S. cerevisiae normally constrain MutLγ to preferentially promote resolution of MutSγ-associated recombination intermediates.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut, United States of America.

ABSTRACT
Accurate chromosome segregation during meiosis relies on the presence of crossover events distributed among all chromosomes. MutSγ and MutLγ homologs (Msh4/5 and Mlh1/3) facilitate the formation of a prominent group of meiotic crossovers that mature within the context of an elaborate chromosomal structure called the synaptonemal complex (SC). SC proteins are required for intermediate steps in the formation of MutSγ-MutLγ crossovers, but whether the assembled SC structure per se is required for MutSγ-MutLγ-dependent crossover recombination events is unknown. Here we describe an interspecies complementation experiment that reveals that the mature SC is dispensable for the formation of Mlh3-dependent crossovers in budding yeast. Zip1 forms a major structural component of the budding yeast SC, and is also required for MutSγ and MutLγ-dependent crossover formation. Kluyveromyces lactis ZIP1 expressed in place of Saccharomyces cerevisiae ZIP1 in S. cerevisiae cells fails to support SC assembly (synapsis) but promotes wild-type crossover levels in those nuclei that progress to form spores. While stable, full-length SC does not assemble in S. cerevisiae cells expressing K. lactis ZIP1, aggregates of K. lactis Zip1 displayed by S. cerevisiae meiotic nuclei are decorated with SC-associated proteins, and K. lactis Zip1 promotes the SUMOylation of the SC central element protein Ecm11, suggesting that K. lactis Zip1 functionally interfaces with components of the S. cerevisiae synapsis machinery. Moreover, K. lactis Zip1-mediated crossovers rely on S. cerevisiae synapsis initiation proteins Zip3, Zip4, Spo16, as well as the Mlh3 protein, as do the crossovers mediated by S. cerevisiae Zip1. Surprisingly, however, K. lactis Zip1-mediated crossovers are largely Msh4/Msh5 (MutSγ)-independent. This separation-of-function version of Zip1 thus reveals that neither assembled SC nor MutSγ is required for Mlh3-dependent crossover formation per se in budding yeast. Our data suggest that features of S. cerevisiae Zip1 or of the assembled SC in S. cerevisiae normally constrain MutLγ to preferentially promote resolution of MutSγ-associated recombination intermediates.

No MeSH data available.


Crossovers mediated by K. lactis Zip1 are dependent on Zip3, Zip4, Spo16 and Mlh3 but independent of Msh4, Msh5 and non-MutSγ-MutLγ crossover pathway components.Sporulating cultures of S. cerevisiae strains carrying one linear and one circular chromosome III and carrying either S. c. ZIP1 (K479), K. l. ZIP1 (K457) or a zip1  (TY521, [18]) allele were embedded in agarose plugs, processed, run on a pulsed-field gel and analyzed by southern blot using a probe to chromosome III sequences (see Methods). Aliquots of sporulating cells were taken at 0, 40, and 70 hours after placement in sporulation medium. (A) shows an example blot that displays bands corresponding to different sized versions of linear chromosome III for the three strains indicated above. Circular chromosomes III present in these strains do not enter the gel. The lowest molecular weight band represents the size of endogenous (linear) III, while the middle and upper bands represent crossover products between linear and circular III; a single crossover event results in a linear chromatid III that runs at the size of the middle band (“dimer”) while a double crossover event involving 3 sister chromatids (of which 2 are circular) produces a “trimer” chromatid III which migrates at the position of the upper band. Plotted on the bar graph below is a value estimating % recombination observed on chromosome III (see Methods) for S. c. ZIP1 (blue), K. l. ZIP1 (green) or zip1  (red) strains at each meiotic time point (see Methods). Open bars displayed by the three graphs in (B) plot the relative % recombination measured for S. c. ZIP1 (blue, top), K. l. ZIP1 (green, middle) or zip1  (red, bottom) strains that are additionally missing the function of a class I or class II meiotic crossover pathway gene (listed on x axis). Each set of three adjacent bars represents samples harvested at 0, 40 and 70 hours (left to right) after placement in sporulation medium. Solid bars at far left of graphs in (B) are the % recombination values for S. c. ZIP1, K. l. ZIP1 or zip1  strains from (A). Graphs plot an average and range for data from at least two independent experiments.
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pgen.1005335.g007: Crossovers mediated by K. lactis Zip1 are dependent on Zip3, Zip4, Spo16 and Mlh3 but independent of Msh4, Msh5 and non-MutSγ-MutLγ crossover pathway components.Sporulating cultures of S. cerevisiae strains carrying one linear and one circular chromosome III and carrying either S. c. ZIP1 (K479), K. l. ZIP1 (K457) or a zip1 (TY521, [18]) allele were embedded in agarose plugs, processed, run on a pulsed-field gel and analyzed by southern blot using a probe to chromosome III sequences (see Methods). Aliquots of sporulating cells were taken at 0, 40, and 70 hours after placement in sporulation medium. (A) shows an example blot that displays bands corresponding to different sized versions of linear chromosome III for the three strains indicated above. Circular chromosomes III present in these strains do not enter the gel. The lowest molecular weight band represents the size of endogenous (linear) III, while the middle and upper bands represent crossover products between linear and circular III; a single crossover event results in a linear chromatid III that runs at the size of the middle band (“dimer”) while a double crossover event involving 3 sister chromatids (of which 2 are circular) produces a “trimer” chromatid III which migrates at the position of the upper band. Plotted on the bar graph below is a value estimating % recombination observed on chromosome III (see Methods) for S. c. ZIP1 (blue), K. l. ZIP1 (green) or zip1 (red) strains at each meiotic time point (see Methods). Open bars displayed by the three graphs in (B) plot the relative % recombination measured for S. c. ZIP1 (blue, top), K. l. ZIP1 (green, middle) or zip1 (red, bottom) strains that are additionally missing the function of a class I or class II meiotic crossover pathway gene (listed on x axis). Each set of three adjacent bars represents samples harvested at 0, 40 and 70 hours (left to right) after placement in sporulation medium. Solid bars at far left of graphs in (B) are the % recombination values for S. c. ZIP1, K. l. ZIP1 or zip1 strains from (A). Graphs plot an average and range for data from at least two independent experiments.

Mentions: In wild-type strains, crossover recombination values estimated using this physical assay for crossovers on chromosome III were at nearly 100% by 40 and 70 hours of sporulation (Fig 7A). In zip1 mutants, on the other hand, approximately 20% and 30% recombination was measured at 40 and 70 hours after transfer to sporulation medium, respectively. In strains expressing K.l. ZIP1, approximately 50% and 65% crossover recombination was measured at 40 and 70 hours of sporulation, respectively (Fig 7A). Because K. l. ZIP1-expressing meiocytes that go on to form spores display wild-type levels of crossing over on chromosome III, the intermediate level of crossing over measured by this physical assay indicates that K. l. ZIP1-expressing meiocytes that fail to form spores are crossover-deficient.


Separable Crossover-Promoting and Crossover-Constraining Aspects of Zip1 Activity during Budding Yeast Meiosis.

Voelkel-Meiman K, Johnston C, Thappeta Y, Subramanian VV, Hochwagen A, MacQueen AJ - PLoS Genet. (2015)

Crossovers mediated by K. lactis Zip1 are dependent on Zip3, Zip4, Spo16 and Mlh3 but independent of Msh4, Msh5 and non-MutSγ-MutLγ crossover pathway components.Sporulating cultures of S. cerevisiae strains carrying one linear and one circular chromosome III and carrying either S. c. ZIP1 (K479), K. l. ZIP1 (K457) or a zip1  (TY521, [18]) allele were embedded in agarose plugs, processed, run on a pulsed-field gel and analyzed by southern blot using a probe to chromosome III sequences (see Methods). Aliquots of sporulating cells were taken at 0, 40, and 70 hours after placement in sporulation medium. (A) shows an example blot that displays bands corresponding to different sized versions of linear chromosome III for the three strains indicated above. Circular chromosomes III present in these strains do not enter the gel. The lowest molecular weight band represents the size of endogenous (linear) III, while the middle and upper bands represent crossover products between linear and circular III; a single crossover event results in a linear chromatid III that runs at the size of the middle band (“dimer”) while a double crossover event involving 3 sister chromatids (of which 2 are circular) produces a “trimer” chromatid III which migrates at the position of the upper band. Plotted on the bar graph below is a value estimating % recombination observed on chromosome III (see Methods) for S. c. ZIP1 (blue), K. l. ZIP1 (green) or zip1  (red) strains at each meiotic time point (see Methods). Open bars displayed by the three graphs in (B) plot the relative % recombination measured for S. c. ZIP1 (blue, top), K. l. ZIP1 (green, middle) or zip1  (red, bottom) strains that are additionally missing the function of a class I or class II meiotic crossover pathway gene (listed on x axis). Each set of three adjacent bars represents samples harvested at 0, 40 and 70 hours (left to right) after placement in sporulation medium. Solid bars at far left of graphs in (B) are the % recombination values for S. c. ZIP1, K. l. ZIP1 or zip1  strains from (A). Graphs plot an average and range for data from at least two independent experiments.
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pgen.1005335.g007: Crossovers mediated by K. lactis Zip1 are dependent on Zip3, Zip4, Spo16 and Mlh3 but independent of Msh4, Msh5 and non-MutSγ-MutLγ crossover pathway components.Sporulating cultures of S. cerevisiae strains carrying one linear and one circular chromosome III and carrying either S. c. ZIP1 (K479), K. l. ZIP1 (K457) or a zip1 (TY521, [18]) allele were embedded in agarose plugs, processed, run on a pulsed-field gel and analyzed by southern blot using a probe to chromosome III sequences (see Methods). Aliquots of sporulating cells were taken at 0, 40, and 70 hours after placement in sporulation medium. (A) shows an example blot that displays bands corresponding to different sized versions of linear chromosome III for the three strains indicated above. Circular chromosomes III present in these strains do not enter the gel. The lowest molecular weight band represents the size of endogenous (linear) III, while the middle and upper bands represent crossover products between linear and circular III; a single crossover event results in a linear chromatid III that runs at the size of the middle band (“dimer”) while a double crossover event involving 3 sister chromatids (of which 2 are circular) produces a “trimer” chromatid III which migrates at the position of the upper band. Plotted on the bar graph below is a value estimating % recombination observed on chromosome III (see Methods) for S. c. ZIP1 (blue), K. l. ZIP1 (green) or zip1 (red) strains at each meiotic time point (see Methods). Open bars displayed by the three graphs in (B) plot the relative % recombination measured for S. c. ZIP1 (blue, top), K. l. ZIP1 (green, middle) or zip1 (red, bottom) strains that are additionally missing the function of a class I or class II meiotic crossover pathway gene (listed on x axis). Each set of three adjacent bars represents samples harvested at 0, 40 and 70 hours (left to right) after placement in sporulation medium. Solid bars at far left of graphs in (B) are the % recombination values for S. c. ZIP1, K. l. ZIP1 or zip1 strains from (A). Graphs plot an average and range for data from at least two independent experiments.
Mentions: In wild-type strains, crossover recombination values estimated using this physical assay for crossovers on chromosome III were at nearly 100% by 40 and 70 hours of sporulation (Fig 7A). In zip1 mutants, on the other hand, approximately 20% and 30% recombination was measured at 40 and 70 hours after transfer to sporulation medium, respectively. In strains expressing K.l. ZIP1, approximately 50% and 65% crossover recombination was measured at 40 and 70 hours of sporulation, respectively (Fig 7A). Because K. l. ZIP1-expressing meiocytes that go on to form spores display wild-type levels of crossing over on chromosome III, the intermediate level of crossing over measured by this physical assay indicates that K. l. ZIP1-expressing meiocytes that fail to form spores are crossover-deficient.

Bottom Line: While stable, full-length SC does not assemble in S. cerevisiae cells expressing K. lactis ZIP1, aggregates of K. lactis Zip1 displayed by S. cerevisiae meiotic nuclei are decorated with SC-associated proteins, and K. lactis Zip1 promotes the SUMOylation of the SC central element protein Ecm11, suggesting that K. lactis Zip1 functionally interfaces with components of the S. cerevisiae synapsis machinery.This separation-of-function version of Zip1 thus reveals that neither assembled SC nor MutSγ is required for Mlh3-dependent crossover formation per se in budding yeast.Our data suggest that features of S. cerevisiae Zip1 or of the assembled SC in S. cerevisiae normally constrain MutLγ to preferentially promote resolution of MutSγ-associated recombination intermediates.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut, United States of America.

ABSTRACT
Accurate chromosome segregation during meiosis relies on the presence of crossover events distributed among all chromosomes. MutSγ and MutLγ homologs (Msh4/5 and Mlh1/3) facilitate the formation of a prominent group of meiotic crossovers that mature within the context of an elaborate chromosomal structure called the synaptonemal complex (SC). SC proteins are required for intermediate steps in the formation of MutSγ-MutLγ crossovers, but whether the assembled SC structure per se is required for MutSγ-MutLγ-dependent crossover recombination events is unknown. Here we describe an interspecies complementation experiment that reveals that the mature SC is dispensable for the formation of Mlh3-dependent crossovers in budding yeast. Zip1 forms a major structural component of the budding yeast SC, and is also required for MutSγ and MutLγ-dependent crossover formation. Kluyveromyces lactis ZIP1 expressed in place of Saccharomyces cerevisiae ZIP1 in S. cerevisiae cells fails to support SC assembly (synapsis) but promotes wild-type crossover levels in those nuclei that progress to form spores. While stable, full-length SC does not assemble in S. cerevisiae cells expressing K. lactis ZIP1, aggregates of K. lactis Zip1 displayed by S. cerevisiae meiotic nuclei are decorated with SC-associated proteins, and K. lactis Zip1 promotes the SUMOylation of the SC central element protein Ecm11, suggesting that K. lactis Zip1 functionally interfaces with components of the S. cerevisiae synapsis machinery. Moreover, K. lactis Zip1-mediated crossovers rely on S. cerevisiae synapsis initiation proteins Zip3, Zip4, Spo16, as well as the Mlh3 protein, as do the crossovers mediated by S. cerevisiae Zip1. Surprisingly, however, K. lactis Zip1-mediated crossovers are largely Msh4/Msh5 (MutSγ)-independent. This separation-of-function version of Zip1 thus reveals that neither assembled SC nor MutSγ is required for Mlh3-dependent crossover formation per se in budding yeast. Our data suggest that features of S. cerevisiae Zip1 or of the assembled SC in S. cerevisiae normally constrain MutLγ to preferentially promote resolution of MutSγ-associated recombination intermediates.

No MeSH data available.