Amy J. MacQueen
During prophase of meiosis I, pairing of homologous chromosomes and interhomolog recombination (crossovers) are essential for proper chromosome segregation. These processes take place in conjunction with the assembly of the Synaptonemal Complex (SC), a conserved tripartite chromosomal structure composed of two lateral elements, a central element, and transverse filaments. The molecular, functional relationship between SC assembly (synapsis) and the critical process of recombination remains obscure. In the budding yeast, Saccharomyces cerevisiae, the major component of the SC transverse filament is the coiled-coil protein, Zip1. Independent of its structural role within the SC, Zip1 also appears to promote crossover activity. Here, we report the results of a large scale structure-function study that homes in on small, adjacent regions within Zip1’s N-terminus that encompass both its synapsis and recombination functions.
The adjacency of Zip1’s pro-synapsis and pro-crossover domains suggests that Zip1 might interact with other SC and/or recombination proteins and facilitate crosstalk between them. However, to date there is insufficient evidence for a direct interaction between Zip1 and any other protein. Based on the observation that zip1-[Δ2-9] and zip1-[Δ10-14] mutant alleles phenocopy cells missing the recombination factor, Zip3, we designed a lacO/LacI corecruitment assay to ask whether Zip1’s N-terminus was sufficient for recruiting Zip3-MYC (or Msh4-MYC, another protein required for recombination) to an ectopic site in the meiotic nucleus. Diploid strains were created in which Zip1’s 348 N-terminal residues were fused to GFP-LacI and that carried a lacO array on chromosome IV. Using immunofluorescence on surface spread meiotic chromosomes, we measured how often Zip3-MYC or Msh4-MYC got recruited to the chimeric Zip1-GFP-LacI protein bound to a lacO array. We conclude that this chimeric version of Zip1 does not consistently recruit Zip3-MYC or Msh4-MYC. These results do not discount the possibility that Zip’s N-terminus directly interfaces with Zip3 or Msh4, but rather suggests a more complex model for how the N-terminus carries out its dual functions.
Additionally, this work recounts the cytological characterization of the DNA helicase Mer3, which has been shown to play an important role in stabilizing recombination intermediates and ensuring that interhomolog crossovers are formed. While Mer3 is often grouped with other proteins (including Zip1, Zip3, and Msh4, among others) that are required for both recombination and synapsis, analysis of its role in SC assembly has been inadequate. Based on analysis of surface spread nuclei, we confirm that mer3Δ mutant cells indeed exhibit a defect in SC assembly, although it retains some ability to build short stretches of SC. Together, these distinct projects contribute to a greater understanding of the rich protein landscape that forms between homologous chromosomes early in meiosis.
Feil, Arden, "Examining the roles and relationships of factors that coordinate chromosome dynamics during meiosis" (2019). Masters Theses. 226.
© Copyright is owned by author of this document