The Identification of a Novel Mutant Allele of topoisomerase II in Caenorhabditis elegans Reveals a Unique Role in Chromosome Segregation During Spermatogenesis

Abstract

Topoisomerase II alleviates DNA entanglements that are generated during mitotic DNA replication, transcription, and sister chromatid separation. In contrast to mitosis, meiosis has two rounds of chromosome segregation following one round of DNA replication. In meiosis II, sister chromatids segregate from each other, similar to mitosis. Meiosis I, on the other hand, segregates homologs, which requires pairing, synapsis, and recombination. The exact role that topoisomerase II plays during meiosis is unknown. In a screen reexamining Caenorhabditis elegans legacy mutants isolated 30 years ago, we identified a novel allele of the gene encoding topoisomerase II, top-2(it7). In this study, we demonstrate that top-2(it7) males produce dead embryos, even when fertilizing wild-type oocytes. Characterization of early embryonic events indicates that fertilization is successful and sperm components are transmitted to the embryo. However, sperm chromatin is not detected in these fertilized embryos. Examination of top-2(it7) spermatogenic germ lines reveals that the sperm DNA fails to segregate properly during anaphase I of meiosis, resulting in anucleate sperm. top-2(it7) chromosome-segregation defects observed during anaphase I are not due to residual entanglements incurred during meiotic DNA replication and are not dependent on SPO-11-induced double-strand DNA breaks. Finally, we show that TOP-2 associates with chromosomes in meiotic prophase and that chromosome association is disrupted in the germ lines of top-2(it7) mutants.

Topoisomerases modulate the timing of meiotic DNA breakage and chromosome morphogenesis inSaccharomyces cerevisiae

“…The meiotic functions of topoisomerases have been primarily elucidated in the context of the meiotic divisions where, similar to mitosis, topo II has a major role in disentangling DNA to facilitate chromosome segregation (Gomez et al, 2014;Hartsuiker et al, 1998;Hughes & Hawley, 2014;Jaramillo-Lambert et al, 2016;Kallio & Lahdetie, 1996;Mengoli et al, 2014;Tateno & Kamiguchi, 2001). However, both topoisomerases are also present and active in meiotic prophase (Borde et al, 1999;Cobb et al, 1997;Stern & Hotta, 1983).…”

“…Only minor meiotic defects have been reported upon inactivation of topo I, including increased gene conversion events in the ribosomal DNA of Saccharomyces cerevisiae (Christman et al, 1988) and mild defects in chromosome pairing in mice (Cobb et al, 1997;Handel et al, 1995). Somewhat more is known about topo II, which localizes diffusely to prophase chromatin in a variety of organisms (Cheng et al, 2006;Cobb et al, 1999;Jaramillo-Lambert et al, 2016;Zhang et al, 2014), with enrichment along chromosome axes noted in some cases (Klein et al, 1992;Moens & Earnshaw, 1989). In Saccharomyces cerevisiae, topo II contributes to proper spacing of crossover events (Zhang et al, 2014).…”

Evaluating alignment and variant-calling software for mutation identification in C. elegans by whole-genome sequencing

“…Paralogs, pseudogenes, and conserved protein-coding domains represent potential sources of sequence duplication that might preclude variant detection in some genes. The impact of sequence duplication is not merely hypothetical: the top-2(it7) mutation cannot be identified by some variant-calling pipelines due to sequence similarity between top-2 and its paralog cin-4 [29]. …”

Cooperation between Caenorhabditis elegansCOMPASS and condensin in germline chromatin organization

“…When temperature-sensitive topoisomerase II mutants are used to bypass its essential requirement in mitosis, defects in chromosome condensation and segregation are also observed in meiosis (Marchetti et al, 2001;Li et al, 2013;Gómez et al, 2014;Hughes & Hawley, 2014). To explore whether set-2 also genetically interacts with top-2 to ensure proper chromosome condensation in the germline, we constructed set-2;top-2 double mutants using a recently described allele, top-2 (it7), that results in a temperature-sensitive chromosome segregation defect in male spermatogenesis (Jaramillo-Lambert et al, 2016).…”