Dynamic functions of DNA2 counteract DNA replication stresses and tumorigenesis

描述(由申请人提供)：细胞对DNA复制压力的反应能力对于保持基因组完整性和防止肿瘤发生至关重要。复制应激既可由内源性因素诱导，也可由环境因素诱导，我们的目标是确定哺乳动物DNA2核酸酶/解旋酶能够缓解不同压力的机制。许多环境因素，如除草剂、羟基脲(HU)、喜树碱(CPT)和电离辐射(IR)等都能引起复制应激，被认为是癌症的致病因素。DNA复制叉停滞不前 如果不稳定和修复，叉子会坍塌，导致双链DNA断裂，导致突变和染色体重排，这是癌前细胞的标志。复制压力也可以由内源性因素引起，例如富含G的序列和难以复制的高度重复的区域。尤其重要的是 端粒是一种特殊的DNA-蛋白质结构，它保护染色体末端免受不适当的降解和融合。哺乳动物的端粒由数百到数千个拷贝的串联重复序列组成，这些重复序列以3‘-单链G突起终止。重复序列是内源性复制压力的来源，因为复制叉会在端粒区域停滞。此外，在复制过程中，3‘G突出部分是无上限的，类似于DNA损伤部位，DNA损伤反应机制可能不适当地针对该部位。保持端粒的完整性是至关重要的；功能失调的端粒通常通过染色体端到端的融合来修复，最终导致多倍体、易位和癌症。酵母中的遗传和分子研究表明，DNA2核酸酶/解旋酶最初被认为只在Okazaki片段成熟的RNA-DNA片断处理中发挥作用，但它参与稳定停滞的DNA复制叉，修复倒塌的DNA复制叉和复制端粒。然而，当我们第一次检查时，我们发现哺乳动物的DNA2蛋白缺乏核定位信号，并定位于线粒体。然而，我们第一个建立的DNA2 KO小鼠模型显示，最显著的表型是端粒缺陷，这意味着DNA2进入细胞核。我们假设DNA2是中和DNA复制压力的主要核酸酶之一。我们的初步数据表明，DNA2蛋白的特定翻译后修饰促进了它在核中的定位，在那里它产生了端粒的3‘G悬垂，保护它免受其他核酸酶的攻击，并抵消其他复制应激，我们的目标是确认这些机制。此外，我们有来自小鼠模型的初步数据显示，即使基因组中一个缺失的DNA2拷贝也会导致高频率的癌症。这是至关重要的信息，首先是因为它不能从酵母研究中推断出来，其次是因为在人类群体和癌症患者中存在自然发生的DNA2 SNPs和突变。我们建议确定这些突变是否使哺乳动物更容易患上癌症。

DESCRIPTION (provided by applicant): The ability of cells to respond to DNA replication stresses is essential for maintaining genomic integrity and preventing tumorigenesis. Replication stress can be induced by both endogenous and environmental factors and our goal is define the mechanisms by which the mammalian DNA2 nuclease/helicase is able to alleviate different stresses. A multitude of environmental agents that are DNA damaging agents such as aphidicolin, hydroxyurea (HU), camptothecin (CPT), and ionized radiation (IR) can cause replication stress are considered as cancer etiological factors. The DNA replication fork stalls at the damage site and if not stabilized and repaired, the fork will collapse resulting in double stranded DNA breaks that lead to mutations and chromosomal rearrangements, a hallmark of pre-cancerous cells. Replication stresses can also arise due to endogenous factors, such as G rich sequences and highly repeated regions that are difficult to replicate. Especially critical are the telomeres, the specialized DNA-protein structures that protect the chromosome ends from inappropriate degradation and fusion. Mammalian telomeres consist of hundreds to thousands of copies of tandem repeats that terminate in a 3' single-stranded G overhang. The repeats are a source of endogenous replication stress, because the replication fork can stall in the telomeric region. Furthermore, during replication the 3' G overhang is uncapped and resembles a site of DNA damage that can be inappropriately targeted by the DNA damage response machinery. Maintaining telomere integrity is essential; dysfunctional telomeres are generally repaired through chromosomal end-to-end fusions that eventually lead to polyploidy, translocations and cancers. Genetic and molecular studies in yeast indicate that the DNA2 nuclease/helicase, initially thought only to play a role in RNA-DNA flap processing during Okazaki fragment maturation, is involved in stabilizing stalled DNA replication forks, repairing collapsed DNA replication forks and replicating the telomeres. However, when first examined, we found the mammalian DNA2 protein lacked a nuclear localization signal and localized to mitochondria. Yet, our first established DNA2 KO mouse model showed that the most significant phenotype is telomere defects, implicating the entrance of DNA2 into the nucleus. We hypothesize DNA2 is one of the primary nucleases that counteracts DNA replication stresses. Our preliminary data that indicates a specific post-translational modification of the DNA2 protein promotes its localization to the nucleus, where it generates the 3' G overhang of the telomere and protects it from attack by other nucleases and counteracts other replication stresses, and we aim to confirm these mechanisms. In addition, we have preliminary data from mouse models showing that even one missing copy of DNA2 from the genome leads to cancers in high frequency. This is critical information, first because it cannot be inferred from yeast studies, and second, because there are naturally occurring DNA2 SNPs and mutations in human populations and cancer patients. We propose to determine if these mutations predispose mammals to cancer development.