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

基本信息

批准号：
10033544
负责人：
Naoko Shima
金额：
$ 30.28万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
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/10033544
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 Fanconi anemia protein Fanconi&apos s Anemia Fibroblasts Future Genes Genetic Genetic Diseases Human Human Genetics Impairment Knowledge Mammalian Cell Mediating Metaphase Mitosis Mitotic Modeling Modification Molecular Monoubiquitination Mus Mutate Nature Normal Cell Oncogenic Pathogenesis Pathway interactions Patients Phenotype Polymerase 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/antagonist mouse model novel novel therapeutics p53-binding protein 1 recruit replication stress ubiquitin-protein ligase

项目摘要

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) 是最近发现的一种现象，在 M 期早期被激活晚期复制中间体 (LRI) 的解析作为支持正确染色体的最终机制有丝分裂期间的分离在受复制应激干扰的 S 期后被强烈诱导。复制叉比正常情况更频繁地出现故障。MiDAS 站点显示为间断站点。前期细胞核中的 DNA 合成，几乎总是在 FANCD2 标记的染色体位点上发现此类位点包括常见的脆弱位点，其中 DNA 复制的完成尤其困难。 MiDAS 的激活在本质上支持此类基因座的稳定性，但它通常会在复制压力下变得困难。可能由于其“最后一刻”的性质，导致中期染色体出现间隙和断裂。认为这种间隙/断裂仍然有助于细胞避免染色体错误分离，这可能导致细胞在最坏的情况下死亡。之前的模型将 RAD52 描述为关键角色，因为它在 MiDAS 的早期阶段发挥作用，并且招募 POLD3，聚合酶 delta 的非催化亚基，对于前期 DNA 合成至关重要。然而，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 MiDAS 没有影响，相反，我们证明 FANCD2 是一种基本调节因子。 MiDAS 的缺陷会非选择性地损害人类细胞中的这一过程。在人类遗传性疾病范可尼贫血 (FA) 中发生突变，我们的发现和初步数据表明 MiDAS 是一种新颖的细胞功能，主要由 FA 基因的子集驱动。我们对哺乳动物细胞中 MiDAS 的修订模型如下：正常人中 MiDAS 的原始形式。细胞本质上是由 FA 驱动机制驱动的，该机制需要 FANCD2 的单泛素化形式 (FANCD2Ub) 和 PCNA (PCNAUb) 可能反映了它们所具有的人类致癌复制应激。与原发性细胞不同，除了 FA 驱动机制外，癌细胞还使用 RAD52 驱动机制。人类细胞中，原代小鼠细胞使用类似于人类癌细胞的两种机制来操作 MiDAS。为了测试这些工作假设，我们提出以下具体目标：1）确定 FA 蛋白的作用在正常人类细胞的 MiDAS 中，2) 确定 PCNAUb 在人类细胞 MiDAS 中的作用，3) 确定 RAD52 驱动的 MiDAS 在人类细胞中的作用，以及 4) 测试小鼠中的 MiDAS 是否同时依赖于 FA 和 RAD52- 该提案的成功完成将揭示 MiDAS 通过不同的方式运行。这些信息将使我们能够正确评估 MiDAS 的作用。各细胞类型的缺陷以供未来研究。