The investigation of paternal-effect lethal mutations in C. elegans

DNA拓扑异构酶是在染色体保真度中发挥关键作用的酶，它通过解开双链DNA中出现的拓扑问题。这种酶主要在有丝分裂中被研究。然而，拓扑异构酶II抑制剂的一个副作用是女性和男性不育，这表明它也在减数分裂细胞周期中起作用。拓扑异构酶II在减数分裂中的确切作用尚不清楚。结合经典遗传学和细胞生物学，我们发现，即使在与野生型卵母细胞受精时，top-2（it7ts）雄性也会产生死胚胎。早期胚胎事件的特征表明受精是成功的，精子成分被传递给胚胎。然而，在这些受精卵中没有检测到精子染色质。对top-2（it7ts）生精细胞系的检查显示，精子DNA在减数分裂过程中不能正常分离。染色质桥在后期I形成，染色质从发育中的精子中移除，产生无核精子。尽管如此，这些无核精子仍然能够受精；早期胚胎事件看起来正常，但胚胎在200个细胞阶段左右停滞。

DNA topoisomerases are enzymes that play a crucial role in chromosome fidelity by disentangling topological problems that arise in double stranded DNA. This enzyme has primarily been studied in mitosis. However, one side effect of topoisomerase II inhibitors is both female and male infertility indicating that it also has functions in the meiotic cell cycle. The exact role that topoisomerase II plays during meiosis is unknown. Using a combination of classical genetics and cell biology, we found that top-2(it7ts) 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(it7ts) spermatogenic germ lines reveals that the sperm DNA fails to segregate properly during the meiotic divisions. Chromatin bridges form during anaphase I and the chromatin is removed from developing spermatids creating anucleate sperm. These anucleate sperm are nonetheless capable of fertilization; early embryonic events appear normal but embryos arrest around the 200-cell stage. Type II DNA topoisomerases have well known roles in DNA replication and chromosome segregation in the mitotic cell cycle of yeast and bacteria. To determine if, similar to other organisms, C. elegans top-2 also plays a role in mitosis, we examined the proliferative zone of top-2(it7ts) males and hermaphrodites where germ cells undergo mitotic proliferation prior to entering meiosis. We found that hermaphrodite and male germ lines have enlarged mitotic germ cells (indicative of an S-phase arrest). In addition, a top-2 non-conditional deletion allele fails to proliferate a germ line. These two results suggest that top-2 has a mitotic function in the proliferative region of the germ line. Given the known enzymatic role of topoisomerase II in disentangling topological problems that arise in double-stranded DNA, we reasoned that TOP-2 might solve topological problems that arise during meiotic crossover recombination. top-2(it7ts) chromosome segregation defects observed during anaphase I are not due to residual entanglements incurred during meiotic DNA replication and are not a consequence of recombination defects. Finally, we tagged TOP-2 with a FLAG epitope by CRISPR/Cas9 gene editing (TOP-2::3XFLAG) to determine TOP-2 localization in the germ lines of hermaphrodites and males. We found that TOP-2 associates with chromosomes in meiotic prophase and that chromosome association is disrupted in the germ lines of top-2(it7ts) mutants. In the future, we aim to use TOP-2::3XFLAG to discover internal phosphorylation and SUMOylation sites: potential post-translational modifications required for TOP-2 regulation. We have also initiated a suppressor screen to identify mutants that restore viability and chromosome segregation to the top-2 mutant. In two trial screens, we have isolated over 10 suppressors that restore viability to over 70%. We are currently carrying out whole genome sequencing to identify the responsible mutation in these strains that is allowing top-2 mutants to grow at non-permissive temperatures. These genes will be the focus of future studies.