Identification of a functional domain in a GADD45-mediated G(2)/M checkpoint

Cell cycle checkpoints are essential for the maintenance of genomic stabili ty in response to DNA damage. We demonstrated recently that GADD45, a DNA d amage-inducible protein, activates a G(2)/M checkpoint induced by either UV radiation or alkylating agents. GADD45 can interact in vivo with the G(2) cell cycle-specific kinase, Cdc2, proliferating cell nuclear antigen (PCNA) , and the cell cycle kinase inhibitor p21(waf1). The ability of GADD45 to i nduce a G2/M arrest may be caused in part by the inhibition of Cdc2 kinase activity. Here, we report the identification of a region of GADD45 that is involved in this G(2)/M checkpoint. Mutants of GADD45 that lacked either th e first 35 or the last 80 residues still retained an ability to induce G(2) /M arrest. A mutant with a deletion of the central region (residues 50-76), which is conserved in the family members GADD45 beta and GADD45 gamma, lac ked such activity. This mutant also lacked an ability to bind to Cdc2, PCNA , and p21(waf1) in vivo. Consistently, either GADD45 beta or GADD45 gamma b ind to Cdc2 in vivo. However, unlike GADD45, neither GADD45 beta nor GADD45 gamma inhibited the Cdc2 kinase or induced G2/M arrest. The unique effect of GADD45 may be caused by the presence of a region containing DEDDDR resid ues. Alanine substitutions in the region abolished GADD45 induction of a G( 2)/M arrest and its inactivation of the Cdc2 kinase but not its binding to Cdc2, PCNA, or p21(waf1). Therefore, the binding of GADD45 to Cdc2 was insu fficient to induce a G2/M arrest, and additional activity contributed by th e DEDDDR residues may be necessary to regulate the G(2)/M checkpoint.