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

线虫父系效应致死突变的研究

• 批准号: 9565922
• 负责人: Andy Golden
• 金额: $ 23.64万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9565922
• 关键词: Adverse effects; Alleles; Anaphase; Bacteria; Binding; CRISPR/Cas technology; Caenorhabditis elegans; Cell Cycle; Cells; Cellular biology; Centrosome; Chromatin; Chromosome Segregation; Chromosomes; DNA; DNA Topoisomerases; DNA biosynthesis; Defect; Development; Diploidy; Embryo; Embryonic Development; Enzymes; Epitopes; Event; Female infertility; Fertilization; Future; Genes; Genetic; Genetic Recombination; Genetic Transcription; Genome; Germ Cells; Germ Lines; Goals; Haploidy; Investigation; Male Infertility; Meiosis; Mitosis; Mitotic; Mitotic Cell Cycle; Mitotic spindle; Morphology; Mutation; Oocytes; Organism; Phosphorylation; Play; Post-Translational Protein Processing; Proliferating; Prophase; Proteins; Regulation; Residual state; Role; S Phase Arrest; Series; Signal Transduction; Site; Spermatids; Temperature; Topoisomerase II; Topoisomerase-II Inhibitor; Yeasts; ds-DNA; genome sequencing; male; mutant; novel; sperm cell; whole genome; zygote

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.

DNA 拓扑异构酶是通过解决双链 DNA 中出现的拓扑问题在染色体保真度中发挥关键作用的酶。这种酶主要在有丝分裂中进行研究。然而，拓扑异构酶 II 抑制剂的副作用之一是导致女性和男性不育，表明它在减数分裂细胞周期中也具有功能。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。结合经典遗传学和细胞生物学，我们发现 top-2(it7ts) 雄性即使在使野生型卵母细胞受精时也会产生死亡胚胎。早期胚胎事件的表征表明受精成功并且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(it7ts) 生精生殖系的检查表明，精子 DNA 在减数分裂期间未能正确分离。染色质桥在后期 I 期间形成，染色质从发育中的精子细胞中去除，形成无核精子。这些无核精子仍然能够受精。早期胚胎事件看似正常，但胚胎在 200 细胞阶段左右停滞。 II 型 DNA 拓扑异构酶在酵母和细菌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。为了确定与其他生物类似，线虫top-2是否也在有丝分裂中发挥作用，我们检查了top-2（it7ts）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雄性生殖细胞有丝分裂生殖细胞增大（表明 S 期停滞）。此外，top-2 非条件删除等位基因无法增殖种系。这两个结果表明top-2在种系的增殖区域具有有丝分裂功能。 鉴于拓扑异构酶 II 在解开双链 DNA 中出现的拓扑问题中已知的酶作用，我们推断 TOP-2 可能会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(it7ts) 染色体分离缺陷不是由于减数分裂 DNA 复制过程中产生的残留缠结，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) 为 TOP-2 标记 FLAG 表位，以确定 TOP-2 在雌雄同体和雄性种系中的定位。我们发现 TOP-2 在减数分裂前期与染色体结合，并且在 top-2(it7ts) 突变体的种系中染色体结合被破坏。未来，我们的目标是使用 TOP-2::3XFLAG 来发现内部磷酸化和 SUMOylation 位点：TOP-2 调节所需的潜在翻译后修饰。我们还启动了抑制子筛选，以鉴定能够恢复 top-2 突变体的活力和染色体分离的突变体。在两次试验筛选中，我们分离出 10 多个抑制剂，它们的活力恢复到 70% 以上。我们目前正在进行全基因组测序，以确定这些菌株中导致 top-2 突变体在不允许的温度下生长的突变。这些基因将是未来研究的重点。