While the ATR-ATRIP complex binds only poorly to RPA complexed with ssDNA lengths of 30 or 50 nt, binding is significantly enhanced in the presence of a 75 nt ssDNA molecule. Complex formation is primarily mediated by physical interaction between ATRIP and RPA. Multiple elements within the ATRIP molecule can bind to the RPA-ssDNA complex, including residues 1-107 (highest affinity), 218-390, and 390-791 (lowest afiinity). Although the full-length ATRIP is unable to bind ssDNA, an internal region (108-390) can weakly bind ssDNA when present in rabbit reticulocyte lysates. ATR can bind to the ssDNA directly independent of RPA, but this binding is inhibited by ATRIP. Upon binding, the ATR kinase becomes activated and can directly phosphorylate substrates such as Rad17.