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.
Our proposal will characterize a mouse model expressing kinase-dead (KD) ATR protein (AtrKD) to determine the kinase-dependent and kinase-independent structural functions of ATR in normal DNA replication, meiosis recombination, and in the face of oncogene-driven replication stresses and genotoxic chemotherapy. The results of these studies will elucidate the role of ATR in DNA damage response and normal DNA replication, and identify the principles and optimal combinations for the use of ATR kinase inhibitors in cancer therapy.
