Genetic mechanisms of Mitotic DNA synthesis in mammalian cells

摘要 有丝分裂DNA合成(MIDAS)是最近发现的一种现象，它在M期早期被激活 晚期复制中间产物(LRIS)的分解是支持正确染色体的最终机制 有丝分裂期间的分离。复制胁迫扰乱S期后强烈诱导MIDAS 复制派生失败的频率比正常情况下更高。Midas的站点显示为带标点符号的站点 早期核的DNA合成，几乎总是在标记FANCD2的染色体上 焦点编队。这些基因座包括常见的脆性位点，其中DNA复制的完成尤其 在复制压力下很难。MIDAS的激活基本上支持了这些基因座的稳定性，但它经常 在中期染色体上造成裂隙和断裂，可能是由于其“最后一分钟”的性质。尽管如此，它还是 认为这样的间隙/断裂仍然有助于细胞避免染色体的错误分离，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 招募POLD3，聚合酶三角洲的一个非催化亚基，它是DNA合成前期必不可少的。 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，因为它缺乏 对正常人体细胞中的MIDAS没有影响。相反，我们展示了FANCD2作为一个基本的调节器 由于它的缺陷，非选择性地损害了人类细胞的这一过程。FANCD2是一种中枢蛋白 突变为一种人类遗传性疾病范可尼贫血(FA)，我们的发现和初步数据表明 MIDAS是一种新的细胞功能，主要由FA基因的子集驱动。 我们对哺乳动物细胞中MIDAS的修正模型如下。正常人MIDAS的原始形态 细胞本质上是由FA驱动的机制驱动的，该机制需要单一泛素化形式的FANCD2 (FANCD2Ub)和增殖细胞核抗原(PCNAUb)。可能反映了它们所蕴含的致癌复制压力，人类 除了FA驱动的机制外，癌细胞还使用RAD52驱动的机制。与主要版本不同 人类细胞、原代小鼠细胞使用与人类癌细胞相似的两种机制运行MIDAS。至 为了验证这些工作假说，我们提出了以下具体目标：1)确定FA蛋白的作用 在正常人细胞的MIDAS中，2)确定PCNAUb在MIDAS中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，以及4)测试小鼠中的MIDAS是否依赖于FA和RAD52- 驱动机构。这项提案的成功完成将揭示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适当地评估MIDAS的影响 各自细胞类型的不足之处有待于今后的研究。

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.