In the activation and maintenance of ATR-dependent DNA damage checkpoint, the interaction between the Rad17-RFC2-5 and 9-1-1 complexes is essential, however, the regulatory mechanism of the interaction is not known. Here we show that vertebrate Rad17 proteins contain a polyanionic 12-amino acid sequence in the C-terminal ends that is important for the 9-1-1 interaction. We demonstrate that the C-terminal tail contains a conserved sequence designated iVERGE that must be intact for the 9-1-1 interaction and contains potential posttranslational modification sites. Our data raise a possibility that the Rad17 C-terminal tail is a molecular switch that regulates the 9-1-1 interaction and the ATR pathway. (C) 2017 Elsevier Inc. All rights reserved.