The structural function of ATR in development, oncogenesis and cancer therapy

项目摘要/摘要 基因组不稳定性驱动癌症的发生、发展和治疗。ATR激酶是一种主要的调节因子 DNA复制和链断裂过程中的DNA损伤反应(DDR)。虽然完全失去了ATR (ATR-/-)废除胚胎发育和细胞活力，ATR激酶抑制剂在 临床前试验，并在与基因毒素联合使用时产生了良好的治疗效果 包括辐射在内的治疗。确定ATR蛋白在DNA复制中是否具有结构功能 和修复(即，独立于其激酶活性或受自动磷酸化调节)，我们产生了一只小鼠 表达激酶死亡ATR蛋白(AtrKD)的模型。ATR+/KD小鼠按预期孟德尔比例出生， 正常大小。此外，使用三苯氧胺诱导的CRE对ATR条件等位基因(AtrC)的体细胞失活 重组酶在AtrC/KD小鼠中耐受性很好，但在AtrC/-小鼠中则不能。而雌性ATR+/KD小鼠是有生育能力的，ATR+/KD小鼠 雄性小鼠由于精子发生过程中的减数分裂缺陷而不育，这在ATR+/-小鼠中是看不到的。在这里我们 建议使用ATR缺失(ATR-)、条件性(AtrC)和激酶死亡(AtrKD)小鼠模型来研究 正常DNA复制和老化过程中的非依赖于激酶的结构功能(目标1)，自动磷酸化 AtrKD蛋白在精子发生过程中的依赖显性负功能(AIM 2)及其致癌作用 以及AtrKD突变对化疗的敏感性。总而言之，这些结果将阐明 ATR在DNA修复和肿瘤发生中的结构功能及其机制 ATR抑制的治疗和副作用。

PROJECT SUMMARY/ABSTRACT Genomic instability drives cancer initiation, progression and treatment. The ATR kinase is a master regulator of DNA damage responses (DDRs) during DNA replication and strand breaks. Although complete loss of ATR (Atr-/-) abrogates embryonic development and cellular viability, ATR kinase inhibitors are well-tolerated in preclinical trials and have generated promising therapeutic effects when used in combination with genotoxic treatments including radiation. To determine whether ATR protein has a structural function in DNA replication and repair (i.e., independent of its kinase activity or regulated by auto-phosphorylation), we generated a mouse model expressing kinase-dead ATR protein (AtrKD). Atr+/KD mice are born at expected Mendelian ratio and of normal size. Moreover somatic inactivation of the Atr conditional allele (AtrC) using Tamoxifen inducible Cre recombinase is very well tolerated in AtrC/KD mice but not AtrC/- mice. While female Atr+/KD mice are fertile, Atr+/KD male mice are sterile due to meiosis defects during spermatogenesis that were not seen in Atr+/- mice. Here we propose to use the Atr null (Atr-), conditional (AtrC), and kinase-dead (AtrKD) mouse models to investigate kinase-independent structural function during normal DNA replication and aging (aim 1), auto-phosphorylation dependent dominant negative function of AtrKD protein during spermatogenesis(aim 2) and the oncogenic potential as well as the chemo-sensitivity profile of the AtrKD mutation. Together, the results will elucidate the structural functions of ATR during DNA repair and oncogenesis, and the mechanisms underlying the therapeutic as well as side effects of ATR inhibition.