Genetic mechanisms of Mitotic DNA synthesis in mammalian cells

摘要 有丝分裂DNA合成（MiDAS）是最近发现的现象，其在早期M期被激活， 晚期复制中间体（LRI）的分解作为支持正确染色体的最终机制 在有丝分裂期间分离。MiDAS在具有复制应激的扰动S期后被强烈诱导，其中 复制分叉失败的频率高于正常情况。MiDAS的部位显示为 在前期细胞核中的DNA合成，其几乎总是在标记有FANCD 2的染色体位点处发现 焦点形成这些基因座包括常见的脆性位点，其中DNA复制的完成特别重要。 在复制压力下很难。MiDAS的激活基本上支持这些基因座的稳定性，但它通常 可能由于其“最后一分钟”的性质，导致中期染色体上的间隙和断裂。但它 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。 先前的模型将RAD 52描述为关键角色，因为它在MiDAS的早期阶段起作用， 招募POLD 3，POLD 3是聚合酶δ的非催化亚基，对前期DNA合成至关重要。 然而，我们最近的研究表明，RAD 52的这种作用仅限于人类癌细胞，因为它的缺乏， 对正常人体细胞中的MiDAS没有影响。相反，我们证明FANCD 2是一个基本的调节器， MiDAS，因为它的缺陷非选择性地损害人类细胞中的这一过程。FANCD 2是一种中心蛋白 在人类遗传性疾病范可尼贫血（FA）中发生突变，我们的发现沿着初步数据表明， MiDAS是一种新的细胞功能，主要由FA基因的子集驱动。 我们在哺乳动物细胞中的MiDAS的修正模型如下。正常人中MiDAS的原始形式 细胞基本上由FA驱动的机制驱动，该机制需要FANCD 2的单泛素化形式 （FANCD 2Ub）和PCNA（PCNAUb）。可能反映了致癌复制的压力，他们港，人类 除了FA驱动的机制之外，癌细胞还使用RAD 52驱动的机制。不同于primary 在人类细胞中，原代小鼠细胞使用与人类癌细胞相似的两种机制来操作MiDAS。到 为了验证这些工作假设，我们提出了以下具体目标：1）确定FA蛋白的作用 2）确定PCNAUb在人细胞中MiDAS中的作用，3）确定PCNAUb在正常人细胞中MiDAS中的作用， RAD 52驱动的MiDAS在人细胞中的作用，以及4）测试小鼠中的MiDAS是否依赖于FA和RAD 52- 驱动机制。该提案的成功完成将揭示MiDAS是通过不同的 在哺乳动物细胞中。这些信息将使我们能够正确评估MiDAS的效果 缺乏相应的细胞类型，用于未来的研究。

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.