Both the basal activity level and TOPBP1-stimulated activity of the S1333A protein are significantly increased compared to the wild type protein. Additionally, S1333 mutations to glycine, arginine, or lysine also create hyperactive kinases. Conversely, a S1333D mutation decreases ATR activity. While we find no evidence that S1333 is phosphorylated in cultured cells, our studies indicate that mutation of a single serine in the large, HEAT repeat region of this 2,644 amino acid protein is sufficient to greatly alter its activity. The exact mechanism mediating this change will require a highresolution structural analysis; however, these mutants provide useful tools for studying the ATR pathway. Our data indicate that a single amino acid change at position 1333, in a region outside of the known regulatory domains, is sufficient to alter ATR kinase activities. In vitro and in cells, S1333A-ATR is hyperactive compared to wild type ATR while S1333D-ATR is less active. Initially, we hypothesized this amino acid is an auto-phosphorylation site regulating ATR kinase activity. However, we were Ro 04-6790 unable to obtain evidence of phosphorylation in cultured cells or in in vitro kinase reactions. Thus, how the mutations alter kinase activity is not clear, but we not known, but HEAT repeats have been shown to serve as protein-protein interaction domains and can also bind DNA. In the structure of DNA-dependent protein kinase, a PIKK family member, the HEAT repeats fold into a double solenoid and form a platform on which the kinase and other C-terminal domains sit. Thus, it is possible that small PSB 36 changes in the HEAT repeat structure are transmitted to the kinase domain, yielding a relatively large and unexpected change in activity. ATRIP also binds to ATR through its HEAT repeats. ATRIP has several functions in ATR signaling including stabilizing the ATR protein, targeting ATR to replication stress sites, and contributing to the interaction with the TOPBP1 protein. TOPBP1 binding to the ATR-ATRIP complex activates ATR by inducing an unknown structural change within ATR that increases ATR substrate affinity. The mutations creating a hyperactive kinase may partly mimic the effect of TOPBP1 binding to ATR-ATRIP and potentiate the ability of TOPBP1 to promote the change in ATR conformation needed for its increased activity. In summary, we identified single amino acid mutations within the ATR HEAT repeats that alter its kinase activity. Cells expressing S1333A-ATR have elevated basal phosphorylation levels of ATR substrates but no noticeable checkpoint or replication defects in cultured cells. Thus, cells can tolerate elevated basal ATR kinase activity. The small decrease in ATR activity caused by the S1333D mutation is enough to cause modest defects in some ATR checkpoint functions. S1333 is not in a region of ATR previously known to be involved in regulation of the kinase.