New roles for Topoisomerase II in meiosis

PROJECT SUMMARY/ABSTRACT Haploid gametes (i.e. eggs and sperm) are generated from diploid precursors through the cell division of meiosis. Eggs and sperm are highly specialized cells that differ in both their morphologies and in their contributions to fertilization and embryogenesis. To achieve these differences, eggs and sperm are generated from meiotic programs with sex-specific characteristics. However, surprisingly little is known about the molecular mechanisms that define sex-specificity. Previously, we identified a novel allele of C. elegans topoisomerase II that uniquely disrupts the segregation of homologous chromosomes during the meiotic divisions of spermatogenesis but not oogenesis. Topoisomerase II (Topo II) is an enzyme that plays a crucial role in chromosome fidelity by disentangling topological problems that arise in double stranded DNA. Topo II is a large ATP-dependent, homodimeric enzyme. Each subunit breaks one DNA strand, passes a second unbroken strand through the break, and then reseals the break. Thus, Topo II enzymes solve topological problems that arise during replication, transcription, chromosome segregation, and recombination. The identification of a sex-specific role for this key, ubiquitous enzyme highlights the differential regulation of the two meiotic programs. Our long-term goal is to understand the molecules and systems that ensure that each egg and sperm receive the correct number of chromosomes during meiosis. To understand this fundamental process, we will utilize the metazoan animal model C. elegans, which, in addition to sexually dimorphic meiotic programs, provides many experimental advantages such as a fast generation time, a transparent body for in vivo analysis of meiosis, and a single top-2 gene. The research in this proposal encompasses two main programs related to sex-specific regulation of meiotic chromosome structure and segregation. Program 1 will identify sex-specific differences in chromosomal axes components, synaptonemal complex (SC) disassembly, and chromosome compaction prior to the segregation of homologous chromosomes during the first meiotic division. Using a targeted candidate gene approach and mutational analysis, including our previously identified sex-specific top-2 allele, we will identify genes that differentially regulate SC disassembly and chromosome compaction in spermatogenesis and oogenesis. Then, in Program 2, we delve into the mechanisms that regulate TOP-2 localization and activity in spermatogenesis and oogenesis. This work builds on our findings that mutations within tyrosyl DNA phosphodiesterase 2 (TDPT-1) can suppress the top-2 mutant phenotypes. Using a combination of biochemical and genetic techniques we will identify the mechanism of suppression of top-2(it7) embryonic lethality for the tdpt-1 mutant suppressors and identify novel TOP-2 interacting proteins during mitosis vs. meiosis and in spermatogenesis vs. oogenesis.