Genetic mechanisms of Mitotic DNA synthesis in mammalian cells

哺乳动物细胞有丝分裂 DNA 合成的遗传机制

• 批准号: 10652323
• 负责人: Naoko Shima
• 金额: $ 31万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10652323
• 关键词: Anaphase; Antimitotic Agents; Aphidicolin; Cell Death; Cell Nucleus; Cell Proliferation; Cell Survival; Cell physiology; Cells; Chromosomal Instability; Chromosomal Stability; Chromosome Fragile Sites; Chromosome Segregation; Chromosomes; Clinical; Closure by clamp; Complex; DNA; DNA Polymerase III; DNA biosynthesis; DNA replication fork; Data; Development; Dose; Embryo; FANCD2 protein; Fanconi anemia protein; Fanconi' s Anemia; Fibroblasts; Future; Genes; Genetic; Genetic Diseases; Human; Human Genetics; Impairment; Knowledge acquisition; Mammalian Cell; Mediating; Metaphase; Mitosis; Mitotic; Modeling; Modification; Molecular; Monoubiquitination; Mus; Mutate; Nature; Normal Cell; Oncogenic; Pathogenesis; Pathway interactions; Patients; Phenotype; Polymerase; Probability; Process; Prometaphase; Prophase; Proteins; RAD52 gene; Resolution; Role; Running; S phase; Sister Chromatid; Site; Slide; Testing; Time; Vertebral column; cancer cell; cell type; chromosome missegregation; inhibitor; micronucleus; mouse model; novel; novel therapeutics; p53-binding protein 1; recruit; replication stress; ubiquitin-protein ligase

Abstract Mitotic DNA synthesis (MiDAS) is a recently discovered phenomenon that is activated in early M phase for the resolution of late replication intermediates (LRIs) as a final mechanism to support proper chromosome segregation during mitosis. MiDAS is strongly induced after perturbed S phase with replication stress where replication forks fail more frequently than normal conditions. Sites of MiDAS appears as punctuated sites of DNA synthesis in prophase nuclei, which are found almost always at chromosome loci marked with FANCD2 focus formation. Such loci include common fragile sites where the completion of DNA replication is particularly difficult under replication stress. Activation of MiDAS essentially supports the stability of such loci, but it often causes gaps and breaks on metaphase chromosomes probably due to its “last minute” nature. Nevertheless, it is considered that such gaps/breaks still help cells avoid chromosome mis-segregation, which may cause cell death in the worst case scenario. A previous model describes RAD52 as a key player, because it functions at an early step of MiDAS and recruits POLD3, a non-catalytic subunit of Polymerase delta, which is essential for DNA synthesis in prophase. However, our recent study revealed that this role of RAD52 is limited to human cancer cells, because its absence has no effect on MiDAS in normal human cells. Rather, we demonstrated FANCD2 as a fundamental regulator of MiDAS, as its deficiency non-selectively impairs this process in human cells. FANCD2 is a central protein mutated in a human genetic disorder Fanconi anemia (FA), and our findings along with preliminary data indicate that MiDAS is a novel cellular function that is primarily driven by a subset of FA genes. Our revised model for MiDAS in mammalian cells is as follows. The original form of MiDAS in normal human cells is essentially driven by the FA-driven mechanism that requires mono-ubiquitinated forms of FANCD2 (FANCD2Ub) and PCNA (PCNAUb). Probably reflecting the oncogenic replication stress that they harbor, human cancer cells use RAD52-driven mechanisms in addition to the FA-driven mechanism. Different from primary human cells, primary mouse cells operate MiDAS using the two mechanisms similar to human cancer cells. To test these working hypotheses, we propose the following specific aims: 1) Determine the roles of the FA proteins in MiDAS in normal human cells, 2) Determine the roles of PCNAUb in MiDAS in human cells, 3) Determine the role of RAD52-driven MiDAS in human cells, and 4) Test if MiDAS in mice depends on both the FA and RAD52- driven mechanisms. Successful completion of this proposal will unveil that MiDAS is run via different mechanisms among mammalian cells. This information will allow us to properly assess the effect of MiDAS deficiency in respective cell types for future studies.

抽象的 有丝分裂DNA合成（MIDAS）是最近发现的现象，在M早期被激活 后期复制中间体（LRI）的分辨率作为支持正确染色体的最终机制 有丝分裂过程中的分离。 MIDA在扰动的S相和复制应力后强烈诱导，其中 复制叉比正常情况更频繁。米达斯的地点似乎是标点的地点 预言核中的DNA合成，几乎总是在标有fancd2的染色体基因座 聚焦形成。这样的地方包括尤其是DNA复制完成的常见脆弱位点 在复制压力下很难。 MIDAS的激活本质上支持了这种地方的稳定性，但通常 导致中期染色体的差距和破裂可能是由于其“最后一分钟”性质。然而，它 被认为这种差距/断裂仍然有助于细胞避免染色体错误脱粒，这可能会导致细胞 在最坏的情况下死亡。 以前的模型将Rad52描述为关键参与者，因为它在MIDAS和MIDAS和 Rectuits Pold3是聚合酶三角洲的非催化亚基，这对于预言中的DNA合成至关重要。 但是，我们最近的研究表明，Rad52的这种作用仅限于人类癌细胞，因为它的缺失 对正常人类细胞中的MIDA没有影响。相反，我们证明了FANCD2是基本的监管机构 MIDAS，由于其缺乏非选择会损害人类细胞中的这一过程。 FANCD2是一种中心蛋白 在人类遗传疾病的fanconi贫血（FA）中突变，我们的发现以及初步数据表明 MIDAS是一种新型细胞功能，主要由FA基因的子集驱动。 我们在哺乳动物细胞中MIDAS的修订模型如下。正常人中Midas的原始形式 细胞基本上是由FA驱动的机制驱动的，该机制需要单泛素化形式的fancd2 （FANCD2UB）和PCNA（PCNAUB）。可能反映了他们所承受的致癌复制应力 除了FA驱动的机制外，癌细胞还使用RAD52驱动的机制。与初级不同 人类细胞，原代小鼠细胞使用类似于人类癌细胞的两种机制运行MIDAS。到 测试这些工作假设，我们提出以下特定目的：1）确定FA蛋白的作用 在正常人细胞中的MIDAS中，2）确定PCNAUB在MIDAS在人类细胞中的作用，3）确定 Rad52驱动的MIDAS在人类细胞中的作用，4）测试小鼠中MIDAS是否取决于FA和Rad52-- 驱动的机制。该提案的成功完成将公布MIDAS通过不同的 哺乳动物细胞之间的机制。这些信息将使我们能够正确评估MIDAS的效果 相对细胞类型的缺乏供以后的研究。