Cooperative roles for Atm and Hus1 in genome maintenance

描述（由申请人提供）：共济失调毛细血管扩张症是一种基因组不稳定综合征，其中DNA损伤检查点蛋白Atm的突变与癌症、不孕症、免疫功能障碍和神经变性有关。Atm在细胞对双链DNA断裂（DSB）的反应中起着核心作用，而涉及相关检查点蛋白Atr的第二种途径对复制应激和大体积DNA损伤以及DSB做出反应。这些通路之间的功能重叠程度尚不清楚，部分原因是Atr通路的成分，包括Chk1和Rad9-Rad1-Hus1（911）复合体，对生存能力至关重要。本申请中描述的研究的长期目标是解决Atm和Atr通路之间的关系，并确定当Atm存在缺陷时Atr通路的活性如何影响疾病的发病机制。研究人员在小鼠中使用了一个Hus1等位基因系列来确定Atm和Atr通路之间的遗传相互作用。当部分Hus1损伤与Atm缺陷合并时，观察到合成致死性，这在两种主要哺乳动物DNA损伤检查点途径之间建立了必要的合作关系。尽管Hus1表达的严重降低与Atm的丧失是致命的，但Hus1表达水平略高的小鼠以低于预期的频率产生活鼠。在Specific Aim 1中，胚胎致死性的基础将通过胚胎的形态学和组织学分析，以及通过检查具有Atm和Hus1缺陷的培养细胞中的DNA损伤信号来确定。此外，同时存在Atm和Hus1缺陷的存活小鼠将进行神经变性测试，这是共济失调毛细血管扩张患者的一种突出表型，在Atm敲除小鼠中未观察到。在Specific Aim 2中，将评估Hus1功能降低对atm缺陷小鼠肿瘤发展的影响。Atm的杂合性增加了人类患乳腺癌的风险，因此，肿瘤的发生也将在具有部分Hus1缺陷的Atm杂合小鼠中进行研究。综上所述，这些研究将阐明两种主要哺乳动物DNA损伤检查点通路之间的关系，并解决这些基因组维持机制在执行发育程序、预防神经变性和抑制肿瘤发生中的合作作用。

DESCRIPTION (provided by applicant): Ataxia Telangiectasia is a genomic instability syndrome in which mutations in the DNA damage checkpoint protein Atm are associated with cancer, infertility, immune dysfunction, and neurodegeneration. Atm plays a central role in cellular responses to double-stranded DNA breaks (DSB), while a second pathway involving the related checkpoint protein Atr responds to replication stress and bulky DNA lesions, as well as DSB. The extent of functional overlap between these pathways is not well understood, in part because components of the Atr pathway, which also includes Chk1 and the Rad9-Rad1-Hus1 (911) complex, are essential for viability. The long-term objectives of the research described in this application are to resolve the relationship between the Atm and Atr pathways, and to determine how the activity of the Atr pathway affects disease pathogenesis when Atm is defective. The investigators employed a Hus1 allelic series in mice to identify genetic interactions between the Atm and Atr pathways. Synthetic lethality was observed when partial Hus1 impairment was combined with Atm deficiency, establishing an essential cooperative relationship between the two primary mammalian DNA damage checkpoint pathways. Although a severe reduction in Hus1 expression was lethal in combination with Atm loss, a slightly higher level of Hus1 expression yielded viable mice at less than expected frequency. In Specific Aim 1, the basis for the embryonic lethality will be determined by morphological and histological analysis of embryos, as well as by examination of DNA damage signaling in cultured cells with both Atm and Hus1 defects. In addition, surviving mice with simultaneous Atm and Hus1 defects will be tested for neurodegeneration, a prominent phenotype of Ataxia Telangiectasia patients that is not observed in Atm knock-out mice. In Specific Aim 2, the impact of reduced Hus1 function on tumor development in Atm-deficient mice will be assessed. Atm heterozygosity confers an increased risk of breast cancer in humans, and therefore tumorigenesis will also be examined in heterozygous Atm mice with a partial Hus1 defect. Taken together, the proposed studies will clarify the relationship between two primary mammalian DNA damage checkpoint pathways and resolve the cooperative roles for these genome maintenance mechanisms in executing developmental programs, preventing neurodegeneration, and suppressing tumorigenesis.