The histone chaperone Asf1 and the checkpoint kinase Rad53 are found in a 
complex in budding yeast cells in the absence of genotoxic stress. Our data 
suggest that this complex involves at least three interaction sites. One site 
involves the H3-binding surface of Asf11 with an as yet undefined surface of 
Rad53. A second site is formed by the Rad53-FHA1 domain binding to Asf1-T(270) 
phosphorylated by casein kinase II. The third site involves the C-terminal 21 
amino acids of Rad53 bound to the conserved Asf1 N-terminal domain. The 
structure of this site showed that the Rad53 C-terminus binds Asf1 in a 
remarkably similar manner to peptides derived from the histone cochaperones HirA 
and CAF-I. We call this binding motif, (R/K)R(I/A/V) (L/P), the AIP box for 
Asf1-Interacting Protein box. Furthermore, C-terminal Rad53-F(820) binds the 
same pocket of Asf1 as does histone H4-F(100). Thus Rad53 competes with histones 
H3-H4 and cochaperones HirA/CAF-I for binding to Asf1. Rad53 is phosphorylated 
and activated upon genotoxic stress. The Asf1-Rad53 complex dissociated when 
cells were treated with hydroxyurea but not methyl-methane-sulfonate, suggesting 
a regulation of the complex as a function of the stress. We identified a rad53 
mutation that destabilized the Asf1-Rad53 complex and increased the viability of 
rad9 and rad24 mutants in conditions of genotoxic stress, suggesting that 
complex stability impacts the DNA damage response.