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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.


Related in: MedlinePlus

S. c. Zip1 and K. l. Zip1 promote Msh4-independent JM formation in S. cerevisiae meiotic cells.Sporulating cultures of S. cerevisiae strains carrying either S. c. ZIP1 (K663), K. l. ZIP1 (K666) or a zip1  (K669) allele in the MSH4 background or S. c. ZIP1 (K672), K. l. ZIP1 (K675) or a zip1  (K678) allele in the msh4 background were subject to psoralen crosslinking to preserve recombination intermediates (JMs; see Methods). Aliquots of sporulating cells were taken at 0, 24, 32, and 40 hours after placement in sporulation medium and crosslinked DNA was separated by 2D gel electrophoresis. In this assay, the linear DNA travels as an arc while branched recombination intermediates (including JMs) are slower migrating and are retarded from the arc of linear fragmented, crosslinked DNA. These molecules can be detected by Southern hybridization as shown in the schematic on the right and images below. The line graphs (A and B) are from a representative time course experiment and plot the percentage of JM/total DNA exhibited by each strain at the ERG1 locus as a function of time. For any given time point, all three strains were analyzed on the same blot; time course experiments were analyzed at least twice with similar trends observed in each experiment. We note that although we do not know the true molecular nature of these JMs, the position of the strong signal–black intermediate labeled “JM” in the schematic—is consistent with that of the dHJ, while the faster migrating signal on the arc of branched molecules (lower grey spot in the schematic) may reflect single-end invasions (SEIs), and the slower migrating signal (higher grey spot in the schematic) could be from multi-chromatid JMs [23,41].
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pgen.1005335.g008: S. c. Zip1 and K. l. Zip1 promote Msh4-independent JM formation in S. cerevisiae meiotic cells.Sporulating cultures of S. cerevisiae strains carrying either S. c. ZIP1 (K663), K. l. ZIP1 (K666) or a zip1 (K669) allele in the MSH4 background or S. c. ZIP1 (K672), K. l. ZIP1 (K675) or a zip1 (K678) allele in the msh4 background were subject to psoralen crosslinking to preserve recombination intermediates (JMs; see Methods). Aliquots of sporulating cells were taken at 0, 24, 32, and 40 hours after placement in sporulation medium and crosslinked DNA was separated by 2D gel electrophoresis. In this assay, the linear DNA travels as an arc while branched recombination intermediates (including JMs) are slower migrating and are retarded from the arc of linear fragmented, crosslinked DNA. These molecules can be detected by Southern hybridization as shown in the schematic on the right and images below. The line graphs (A and B) are from a representative time course experiment and plot the percentage of JM/total DNA exhibited by each strain at the ERG1 locus as a function of time. For any given time point, all three strains were analyzed on the same blot; time course experiments were analyzed at least twice with similar trends observed in each experiment. We note that although we do not know the true molecular nature of these JMs, the position of the strong signal–black intermediate labeled “JM” in the schematic—is consistent with that of the dHJ, while the faster migrating signal on the arc of branched molecules (lower grey spot in the schematic) may reflect single-end invasions (SEIs), and the slower migrating signal (higher grey spot in the schematic) could be from multi-chromatid JMs [23,41].

Mentions: We examined the capacity for K. l. Zip1 to facilitate MutSγ-independent recombination in greater detail by asking whether K. l. Zip1 can rescue the JM deficit reported for cells missing MutSγ complex function [27]. We analyzed six strains, each carrying S.c. ZIP1, K.l. ZIP1 or a zip1 allele, in either a MSH4 or a msh4 background. As our strains (BR1919-8B-derived [69]) progress through meiosis in an asynchronous manner, we reasoned that we would be more likely to detect JMs if we prevent their resolution. Thus, each of our strains is also missing NDT80 activity, which is normally required to promote the molecular pathways that resolve JMs into crossovers in S. cerevisiae [3,19], and is indeed required for crossover formation in K. l. ZIP1-expressing cells (S6 Fig). Cells were harvested at 0, 24, 32 and 40 hours after being introduced into sporulation medium, then subjected to psoralen crosslinking to preserve JM structures. Crosslinked DNA was extracted, digested with HindIII, and DNA fragments were separated by two-dimensional (2D) electrophoresis. The branched nature of crosslinked JMs causes them to migrate to a position on the 2D gel which is displaced from the arc of the bulk of crosslinked genomic DNA [5,35] (see cartoon in Fig 8). The positions of all DNA fragments that correspond to the ERG1 and YCR047c loci, which are associated with DSB hotspots [35,77,80,81], were analyzed by Southern blot hybridization.


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)

S. c. Zip1 and K. l. Zip1 promote Msh4-independent JM formation in S. cerevisiae meiotic cells.Sporulating cultures of S. cerevisiae strains carrying either S. c. ZIP1 (K663), K. l. ZIP1 (K666) or a zip1  (K669) allele in the MSH4 background or S. c. ZIP1 (K672), K. l. ZIP1 (K675) or a zip1  (K678) allele in the msh4 background were subject to psoralen crosslinking to preserve recombination intermediates (JMs; see Methods). Aliquots of sporulating cells were taken at 0, 24, 32, and 40 hours after placement in sporulation medium and crosslinked DNA was separated by 2D gel electrophoresis. In this assay, the linear DNA travels as an arc while branched recombination intermediates (including JMs) are slower migrating and are retarded from the arc of linear fragmented, crosslinked DNA. These molecules can be detected by Southern hybridization as shown in the schematic on the right and images below. The line graphs (A and B) are from a representative time course experiment and plot the percentage of JM/total DNA exhibited by each strain at the ERG1 locus as a function of time. For any given time point, all three strains were analyzed on the same blot; time course experiments were analyzed at least twice with similar trends observed in each experiment. We note that although we do not know the true molecular nature of these JMs, the position of the strong signal–black intermediate labeled “JM” in the schematic—is consistent with that of the dHJ, while the faster migrating signal on the arc of branched molecules (lower grey spot in the schematic) may reflect single-end invasions (SEIs), and the slower migrating signal (higher grey spot in the schematic) could be from multi-chromatid JMs [23,41].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482702&req=5

pgen.1005335.g008: S. c. Zip1 and K. l. Zip1 promote Msh4-independent JM formation in S. cerevisiae meiotic cells.Sporulating cultures of S. cerevisiae strains carrying either S. c. ZIP1 (K663), K. l. ZIP1 (K666) or a zip1 (K669) allele in the MSH4 background or S. c. ZIP1 (K672), K. l. ZIP1 (K675) or a zip1 (K678) allele in the msh4 background were subject to psoralen crosslinking to preserve recombination intermediates (JMs; see Methods). Aliquots of sporulating cells were taken at 0, 24, 32, and 40 hours after placement in sporulation medium and crosslinked DNA was separated by 2D gel electrophoresis. In this assay, the linear DNA travels as an arc while branched recombination intermediates (including JMs) are slower migrating and are retarded from the arc of linear fragmented, crosslinked DNA. These molecules can be detected by Southern hybridization as shown in the schematic on the right and images below. The line graphs (A and B) are from a representative time course experiment and plot the percentage of JM/total DNA exhibited by each strain at the ERG1 locus as a function of time. For any given time point, all three strains were analyzed on the same blot; time course experiments were analyzed at least twice with similar trends observed in each experiment. We note that although we do not know the true molecular nature of these JMs, the position of the strong signal–black intermediate labeled “JM” in the schematic—is consistent with that of the dHJ, while the faster migrating signal on the arc of branched molecules (lower grey spot in the schematic) may reflect single-end invasions (SEIs), and the slower migrating signal (higher grey spot in the schematic) could be from multi-chromatid JMs [23,41].
Mentions: We examined the capacity for K. l. Zip1 to facilitate MutSγ-independent recombination in greater detail by asking whether K. l. Zip1 can rescue the JM deficit reported for cells missing MutSγ complex function [27]. We analyzed six strains, each carrying S.c. ZIP1, K.l. ZIP1 or a zip1 allele, in either a MSH4 or a msh4 background. As our strains (BR1919-8B-derived [69]) progress through meiosis in an asynchronous manner, we reasoned that we would be more likely to detect JMs if we prevent their resolution. Thus, each of our strains is also missing NDT80 activity, which is normally required to promote the molecular pathways that resolve JMs into crossovers in S. cerevisiae [3,19], and is indeed required for crossover formation in K. l. ZIP1-expressing cells (S6 Fig). Cells were harvested at 0, 24, 32 and 40 hours after being introduced into sporulation medium, then subjected to psoralen crosslinking to preserve JM structures. Crosslinked DNA was extracted, digested with HindIII, and DNA fragments were separated by two-dimensional (2D) electrophoresis. The branched nature of crosslinked JMs causes them to migrate to a position on the 2D gel which is displaced from the arc of the bulk of crosslinked genomic DNA [5,35] (see cartoon in Fig 8). The positions of all DNA fragments that correspond to the ERG1 and YCR047c loci, which are associated with DSB hotspots [35,77,80,81], were analyzed by Southern blot hybridization.

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.


Related in: MedlinePlus