Construction of His-tagged ATR fusion proteins: working toward the identification of ATR-interacting proteins

Abstract

The protein kinase ATR is responsible for initiating signal transmission at cell cycle checkpoints. ATR is a 303 kDa protein containing a carboxy-terminus kinase domain related to PI-3 kinase, and is homologous to the ATM gene product in human cells and the rad3/MEC1 proteins in yeast. These proteins, together with DNA-PK, are part of the family of PI-3 kinase-related kinases (PIKKs). All members of this family play important roles in cell cycle checkpoints and operate to permit cell survival following many forms of DNA damage. Upon damage of DNA in eukaryotic cells, ATR undergoes a dramatic intranuclear relocalization, translocating to nuclear foci that represent sites of DNA damage and repair. The ultimate goals of this research are to identify new checkpoint pathway substrates that ATR is able to phosphorylate post-DNA damage, and to initiate the identification of potential inhibitors of ATR function. This study commenced by dividing ATR into seven fragments. These fragments were subsequently cloned into a vector system and transformed in order to generate ATR fusion proteins. The induction of these fusion constructs, mediated by their screening and conditioning, is subject to a wide variety of specifications. Because the optimal induction conditions have yet to be discovered, fine-tuning of the experimental techniques must continue in order to give this research a higher degree of significance. However, once successfully induced, a series of cotransfection experiments may be initiated, in which the activity of ATR, plus several additional proteins can be monitored under condition of DNA damage.