Regulation of re-replication in mammalian cells

项目摘要 哺乳动物细胞进化出多种不重叠的机制，以确保DNA复制启动 在每个分裂周期中只有一次复制的起源；失去对这些机制的控制 导致基因组不稳定，这是恶性转化的重要驱动因素。越来越多的证据表明 高等真核生物的起源、利用和激活受到表观遗传因素的影响，但确切的机制 在很大程度上是不确定的。我们的长期目标是阐明控制复制的基础机制 在哺乳动物细胞中的启动并理解这些机制的扰动如何引发基因组 不稳定。组蛋白甲基转移酶Set8正在成为复制启动的关键调节因子 哺乳动物细胞通过其对组蛋白H4K20的单甲基转移酶活性。细胞周期调节酶 在细胞周期的G1期对起源许可是必不可少的，但在S期被蛋白质降解；阻断 这一步骤触发了相同细胞周期内的重复复制启动或再复制。集8和 然而，H4K20me也参与转录抑制和DNA双链断裂的修复 (DSB)，但这些看似独立的活动是否在复制启动或重新复制中发挥作用 不知道。最重要的是，人们对重新复制的性质或特征知之甚少 在有缺陷的Set8降解的单元中积累的产品，也没有关于 发生基因组重新复制，或者如果存在更容易重新复制的某些基因组区域 归纳法。我们的新结果表明，由有缺陷的Set8降解引起的再复制不是随机的 具有较少基因组位置的过程显示出大量且显著的拷贝数增加，这让人联想到基因组 在癌细胞中看到的放大。其他初步研究表明，重新复制可能会 起源于DNA双链断裂(DSB)，它可能在复制过程中自发出现，需要 参与转录沉默和DSB修复的基因的活性。我们创新的初步方案 研究和实验方法旨在彻底检查这种替代的重复复制模式 归纳法。目的1我们将确定Re基因的大小(拷贝数增益)和基因组分布。 在有缺陷的Set8降解的细胞中复制DNA，并在DSB诱导之后。我们还将测试是否 这些参数在不同的癌细胞类型以及癌症和非癌症细胞中是不同的。目标2将决定 异常稳定的Set8和甲基化的H4K20在Set8缺陷细胞中的染色质占有率 降解，以及这些区域是否与重新复制的DNA区域重叠。目标3将定义 转录抑制和DSB修复蛋白在再复制诱导中的作用。成功地执行了 提出的AIMS有望增加我们对 并有助于更好地理解这些机制的扰动是如何引发 基因组不稳定。

Project Summary Mammalian cells have evolved multiple non-overlapping mechanisms to ensure that DNA replication initiates from origins of replications once and only once in each division cycle; loss of control over these mechanisms induces genomic instability, an important driver of malignant transformation. Increasing evidence suggests that origin utilization and activation in higher eukaryotes is influenced by epigenetic factors, but exact mechanisms are largely undefined. Our long-term goals are to elucidate the underpinning mechanisms that control replication initiation in mammalian cells and to understand how perturbations of these mechanisms provokes genomic instability. The histone methyltransferase SET8 is emerging as a key regulator of replication initiation in mammalian cells through its mono-methyltransferase activity on histone H4K20. The cell cycle regulated enzyme is essential for origin licensing in G1 phase of the cell cycle, but is proteolytically degraded in S-phase; blocking this step triggers reiterative replication initiation within the same cell cycle or re-replication. Both SET8 and H4K20me, however, are also involved in transcriptional repression and in the repair of DNA double strand breaks (DSBs), but whether these seemingly independent activities play a role in replication initiation or re-replication is not known. Most importantly, little to nothing is known about the nature or characteristics of the re-replication products that accumulate in cells with defective SET8 degradation, nor is there information on where in the genome re-replication occurs or if there are certain genomic regions that are more prone to re-replication induction. Our new results show that re-replication resulting from defective SET8 degradation is not a stochastic process with few genomic sites exhibit large and significant copy number gains, reminiscent of genomic amplifications that are seen in cancer cells. Additional preliminary studies suggest that re-replication may originate from DNA double strand breaks (DSBs) that may spontaneously arise during replication, and requires the activity of genes involved both in transcriptional silencing and in DSB repair. Our innovative preliminary studies and experimental approaches are designed to thoroughly examine this alternative model of re-replication induction. Aim 1 we will determine the magnitude (copy number gains) and genomic distribution of the re- replicated DNA in cells with defective SET8 degradation and following the induction of DSBs. We will also test if these parameters vary in different cancer cell types and in cancer vs. non-cancer cells. Aim 2 will determine the chromatin occupancy of aberrantly stabilized SET8 and methylated H4K20 in cells with defective SET8 degradation, and whether these overlap with regions of re-replicated DNA. Aim 3 will define the role of transcriptional repression and DSB repair proteins in re-replication induction. The successful execution of the proposed aims promises to increase our understanding of the mechanisms regulating replication initiation in mammalian cells, and lead to a better understanding of how perturbations of these mechanisms provokes genomic instability.