Abstract
Forkhead-associated (FHA) domains are phosphothreonine-binding modules prevalent in proteins with important cell cycle and DNA damage response functions. The yeast checkpoint kinase Rad53 is unique in containing two FHA domains. We have generated novel recessive rad53 alleles with abolished FHA domain functions resulting from Ala substitution of the critical phosphothreonine-binding residues Arg70 and Arg605. In asynchronous cells, inactivation of the N-terminal FHA1 domain did not impair Rad53 activation and downstream functions, whereas inactivation of the C-terminal FHA2 domain led to reduced Rad53 activation and significantly increased DNA damage sensitivity. Simultaneous inactivation of both FHA domains abolished Rad53 activation and all downstream functions and dramatically increased the sensitivity to DNA damage and replication blocks similar to kinase-defective and rad53 null alleles, but did not compromise the essential viability function of Rad53. Interestingly, in G2/M synchronized cells, mutation of either FHA domain prevented Rad53 activation and impaired the cell cycle arrest checkpoint. Our data demonstrate that both FHA domains are required for normal Rad53 functions and indicate that the two FHA domains have differential but partially overlapping roles in Rad53 activation and downstream signaling.
Highlights
The finding that the C-terminal FHA2 domain plays an important role in the DNA damage-dependent activation of Rad53 by binding to the phosphorylated upstream protein Rad9 was instrumental in defining FHA domains as Thr(P)binding modules [8]
The results presented here demonstrate that the two FHA domains play important roles in activation of the Rad53 kinase in response to DNA damage
The two FHA domains seem to contribute differentially to Rad53 activation depending on the cell cycle stage
Summary
Forkhead-associated (FHA) domains are phosphothreonine-binding modules prevalent in proteins with important cell cycle and DNA damage response functions. Inactivation of the N-terminal FHA1 domain did not impair Rad activation and downstream functions, whereas inactivation of the C-terminal FHA2 domain led to reduced Rad activation and significantly increased DNA damage sensitivity. The finding that the C-terminal FHA2 domain plays an important role in the DNA damage-dependent activation of Rad by binding to the phosphorylated upstream protein Rad was instrumental in defining FHA domains as Thr(P)binding modules [8]. Other proposed Rad FHA domain ligands include Dbf that can interact in yeast twohybrid and in vitro assays with both the FHA1 and FHA2 domain [12] and the protein phosphatases Ptc and Ptc that can interact with the FHA1 domain and down-regulate Rad activity in the recovery from DNA damage [13]. An important question considering the presence of two FHA domains in Rad compared with a single N-terminal FHA domain in other related kinases is: do the two FHA domains have independent specialized functions, or do they act in a redundant manner?
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