The human ATR-mediated DNA damage checkpoint in a reconstituted system.

DNA damage checkpoints are signal transduction pathways that coordinate the cell cycle with other cellular responses to DNA damage in order to maintain genomic integrity. The ATR kinase is responsible for initiating the DNA damage checkpoint in response to UV-damaged DNA and incompletely replicated DNA. We have recently established an in vitro system that recapitulates most but not all features of the human ATR-mediated DNA damage checkpoint response. With this system we have shown that damaged DNA can be a direct signal for ATR activation, and that TopBP1 specifically stimulates ATR in the presence of damaged DNA under physiological ionic strength conditions. This system provides a powerful tool to gain insight into the molecular mechanism of the ATR pathway. Here we describe preparation of the checkpoint components and our specific kinase assay in more detail.