Maintenance of Genome Stability and Breast Cancer: Molecular Analysis of DNA Damage-Activated Kinases

摘要

Genomic instability is a hallmark of breast cancer cells. DNA damage checkpoints are critical for the prevention of genomic instability and breast cancer. The ATR checkpoint kinase is activated in response to exogenous and endogenous DNA damage, and phosphorylates downsteam substrates such as BRCA1 and p53 to promote cell cycle arrest DNA repair, and apoptosis. ATR exists in a complex with ATR-interacting protein (ATRIP). In response to DNA damage, the ATR-ATRIP complex is recruited to DNA lesions in part through an interaction between ATRIP and the single-stranded DNA binding protein RPA (Replication Protein A). We report the identification of a conserved checkpoint protein recruitment domain (CRD) in ATRIP orthologs by biochemical mapping of the RPA binding site in combination with NMR, mutagenesis, and computational modeling studies. Mutations in the CRD of the yeast ATRIP ortholog Ddc2 disrupt the Ddc2- RPA interaction, prevent proper localization of Ddc2 to DNA breaks, sensitize yeast to DNA damaging agents, and partially compromise checkpoint signaling. We have also defined a conserved TopBP1 interacting region in ATRIP that is necessary for ATR activation. Finally, we have discovered a PIKK regulatory domain (PRD) in ATR that is required for its activation by TopBP1 and cellular viability. Thus, our results support a multi-step model for ATR activation that requires separable localization and activation functions of ATRIP, and helps to explain how cells maintain genome stability and prevent tumorigenesis.