Reconstitution of a human ATR-mediated DNA damage checkpoint response

The genomes of eukaryotic cells are constantly under assault by exogenous and endogenous forms of DNA damage. In response to DNA damage, cells activate the DNA damage checkpoint response which delays cell cycle progression. Accumulating evidence indicates that genetic checkpoint defects lead to pronounced predisposition to cancer. ATR is a key regulator of the UV-induced DNA damage checkpoint response and activates the downstream Chk1 protein kinase, which in turn affects cell cycle regulatory proteins. Despite recent progress in characterizing the molecular components of the ATR-mediated checkpoint pathway, how the components work coordinately in response to DNA damage remains ill-defined. To understand the molecular mechanism of this checkpoint response, it is necessary to develop an in vitro checkpoint system reconstituted from purified proteins. Therefore, I describe here an in vitro system with purified human checkpoint proteins that recapitulates key elements of the ATR-mediated DNA damage checkpoint. The data from this system show that damaged DNA specifically stimulates TopBP1-dependent activation of ATR kinase activity toward its substrates, Chk1 and p53. Moreover, the in vitro system provides a useful tool for studying the DNA binding and ATR stimulatory activities of TopBP1 and indicates a cooperative activation mechanism for ATR activation. Finally, I demonstrate in vitro evidence indicating the stimulatory effect of RPA-coated ssDNA on TopBP1- dependent ATR activation. I have established a useful in vitro system for studying stimulation of ATR kinase activity by other checkpoint components and provide insight into the molecular mechanism of the ATR-mediated checkpoint response. Moreover, my work will eventually contribute to the reconstitution of the complete in vitro checkpoint response encompassing all identified checkpoint components