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

DNA拓扑异构酶是通过解开双链DNA中出现的拓扑问题在染色体保真度中起关键作用的酶。这种酶主要在有丝分裂中进行研究。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，表明它在减数分裂细胞周期中也有功能。拓扑异构酶II在减数分裂过程中发挥的确切作用尚不清楚。通过结合经典遗传学和细胞生物学，我们发现，前2名（it 7 ts）雄性产生死亡胚胎，即使是野生型卵母细胞。早期胚胎事件的特征表明受精是成功的，精子成分被传递到胚胎。然而，在这些受精胚胎中未检测到精子染色质。对top-2（it 7 ts）生精生殖系的研究表明，精子DNA在减数分裂过程中不能正确分离。染色质桥在后期I形成，染色质从发育中的精子细胞中去除，产生无核精子。这些无核精子仍然能够受精;早期胚胎事件似乎正常，但胚胎在200个细胞阶段左右停止。 II型DNA拓扑异构酶在酵母和细菌的有丝分裂细胞周期中的DNA复制和染色体分离中具有众所周知的作用。为了确定C.线虫TOP-2也在有丝分裂中起作用，我们检查了TOP-2（IT 7 TS）雄性和雌雄同体的增殖区，其中生殖细胞在进入减数分裂之前经历有丝分裂增殖。我们发现，雌雄同体和男性生殖细胞系有扩大的有丝分裂生殖细胞（S期阻滞的指示）。此外，前2个非条件性缺失等位基因不能增殖生殖系。这两个结果表明，top-2在生殖细胞系的增殖区域具有有丝分裂功能。 鉴于已知的拓扑异构酶II在解开双链DNA中出现的拓扑问题中的酶促作用，我们推断TOP-2可能解决减数分裂交叉重组过程中出现的拓扑问题。在后期I期间观察到的top-2（it 7 ts）染色体分离缺陷不是由于减数分裂DNA复制期间发生的残余缠结，也不是重组缺陷的结果。 最后，我们通过CRISPR/Cas9基因编辑（TOP-2：：3XFLAG）用FLAG表位标记TOP-2，以确定TOP-2在雌雄同体和雄性生殖系中的定位。我们发现TOP-2与减数分裂前期的染色体相关联，并且在TOP-2（it 7 ts）突变体的生殖系中染色体关联被破坏。在未来，我们的目标是使用TOP-2：：3XFLAG来发现内部磷酸化和SUMO化位点：TOP-2调控所需的潜在翻译后修饰。我们还进行了抑制筛选，以确定恢复活力和染色体分离的前2位突变体的突变体。

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 also are conducting a suppressor screen to identify mutants that restore viability and chromosome segregation to the top-2 mutant.