Genome maintenance by the mouse Hus1 checkpoint gene

描述(由申请人提供)：细胞持续遭受基因组损伤，如果没有得到适当的治疗，可能会导致发育缺陷、过早衰老和癌症易感性增加。DNA损伤检查点通过调节细胞周期转变、细胞凋亡和DNA修复来防御基因组不稳定的后果。在哺乳动物中，检查点蛋白Hus1是响应复制压力和各种DNA损伤的途径的关键组成部分。HUS1依赖的途径对于胚胎发育是必不可少的，因此它在动物中的许多生物学功能还不是很清楚。这项研究的长期目标是了解依赖Hus1的检查点通路如何保护基因组的完整性，并确定这一机制中的缺陷如何影响肿瘤发生和生理DNA损伤反应。拟议的研究将通过对小鼠Hus1基因组位点进行独特的靶向修饰来推进这些重要的目标。目的研究HUS1的部分或完全失活对癌基因激活诱导的肿瘤发生的影响，以解决HUS1作为候选抑癌基因和癌细胞存活和增殖所需的相反作用。在第二个目标中，体内DNA损伤反应中对Hus1的需求将通过测量Hus1表达减少的小鼠的组织特异性遗传毒性应激反应来确定，部分原因是为了获得关于检查点功能如何影响对DNA损伤抗癌治疗的敏感性的新见解。这些对前两个目标中检查点功能障碍的生理后果的分析将得到目标三中HUS1分子机制的研究的补充，以努力定义对于遗传毒性应激生存至关重要的HUS1功能，并确定HUS1序列变异如何影响基因组维持。总之，拟议的研究将定义必要的检查点机制如何在肿瘤发生和组织特异性DNA损伤反应中发挥作用，并将为自发突变、自然多态或药物抑制引起的检查点功能障碍的生物医学意义提供见解。公共卫生相关性：DNA损伤检查点通过防御基因组的破坏，对于正常的胚胎发育、对环境压力和DNA破坏性化疗的保护性反应以及成年生物的无肿瘤生存至关重要。这一方案的实验利用新的基因工程小鼠品系来分析必要的DNA损伤检查点基因Hus1的生物学功能。这些研究为更好地了解癌症是如何发展的，正常细胞和肿瘤细胞如何对抗癌治疗做出反应，以及这些过程如何受到Hus1序列变异(包括自发突变和自然多态)的影响带来了巨大的希望。

DESCRIPTION (provided by applicant): Cells continuously experience genome damage that, if not properly attended to, can cause developmental defects, premature aging, and increased cancer predisposition. DNA damage checkpoints defend against these consequences of genomic instability by regulating cell cycle transitions, apoptosis, and DNA repair. In mammals, the checkpoint protein Hus1 is a critical component of a pathway that responds to replication stress and a variety of DNA lesions. The Hus1- dependent pathway is essential for embryonic development, and consequently many of its biological functions in animals are not well-defined. The long-term goals of the research described in this proposal are to understand how the Hus1-dependent checkpoint pathway protects genomic integrity and to determine how defects in this mechanism affect tumorigenesis and physiological DNA damage responses. The proposed studies will advance these important objectives using a unique collection of targeted modifications at the mouse Hus1 genomic locus. Aim one addresses how partial or complete Hus1 inactivation affects tumor development induced by activated oncogenes in order to resolve the opposing roles of Hus1 as a candidate tumor suppressor and a factor required for cancer cell survival and proliferation. In Aim Two, the requirements for Hus1 during in vivo DNA damage responses will be determined by measuring the tissue-specific genotoxic stress responses of mice with reduced Hus1 expression, in part to gain new insights into how checkpoint function impacts sensitivity to DNA damaging anti-cancer therapies. These analyses of the physiological consequences of checkpoint dysfunction in the first two aims will be complemented by the investigation of Hus1 molecular mechanisms in Aim Three, in an effort to define the Hus1 functions that are essential for the survival of genotoxic stress and to establish how Hus1 sequence variations affect genome maintenance. Together, the proposed studies will define how an essential checkpoint mechanism functions during tumorigenesis and tissue-specific DNA damage responses, and will provide insights into the biomedical implications of checkpoint dysfunction stemming from spontaneous mutations, natural polymorphisms, or pharmacological inhibition. PUBLIC HEALTH RELEVANCE: By defending against damage to the genome, DNA damage checkpoints are critical for normal embryonic development, protective responses to environmental stresses and DNA damaging chemotherapeutics, and the tumor-free survival of adult organisms. The experiments of this proposal make use of novel genetically-engineered mouse strains to analyze the biological functions of the essential DNA damage checkpoint gene Hus1. These studies hold great promise for improving the understanding of how cancers develop and how normal and neoplastic cells respond to anti-cancer therapies, as well as how these processes are influenced by Hus1 sequence variations, including spontaneous mutations and natural polymorphisms.