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

项目总结/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（DDRs）。虽然ATR完全丧失 (Atr-/-）消除胚胎发育和细胞活力，ATR激酶抑制剂在哺乳动物中耐受良好。 临床前试验，并产生了有希望的治疗效果时，与遗传毒性组合使用 治疗包括放射治疗。为了确定ATR蛋白在DNA复制中是否具有结构功能， 并修复（即，独立于其激酶活性或受自身磷酸化调节），我们产生了小鼠 表达激酶死亡ATR蛋白（AtrKD）的模型。Atr +/KD小鼠以预期的孟德尔比率出生， 正常大小。此外，使用他莫昔芬诱导的Cre对Atr条件等位基因（AtrC）进行体细胞失活， 重组酶在AtrC/KD小鼠中耐受性非常好，但在AtrC/-小鼠中不是。虽然雌性Atr +/KD小鼠是可生育的，但Atr +/KD 雄性小鼠由于在精子发生期间的减数分裂缺陷而不育，这在Atr +/-小鼠中未观察到。这里我们 我建议使用Atr null（Atr-）、条件性（AtrC）和激酶死亡（AtrKD）小鼠模型来研究 正常DNA复制和老化过程中激酶非依赖性结构功能（目的1），自磷酸化 AtrKD蛋白在精子发生过程中的依赖性显性负功能（aim 2）和致癌性 潜在的以及AtrKD突变的化学敏感性概况。总之，这些结果将阐明 ATR在DNA修复和肿瘤发生过程中的结构功能，以及其潜在的机制， 治疗以及ATR抑制的副作用。

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.