Characterization of the activation domain of the Rad53 checkpoint kinase.

Rad53 protein, the yeast orthologue of the human checkpoint kinase Chk2, presents two highly conserved phosphorylatable threonine residues (T354 and T358) in the activation domain, whose phosphorylation is critical to allow the activation of the kinase. In this study we found that Rad53 protein variants in which alanine and/or aspartate replace the threonine residues 354 and/or 358 do not retain kinase activity and do not undergo autophosphorylation, leading to defect in the checkpoint response and iper-sensitivity to DNA damage and DNA replication stress agents. Interestingly, we found that the rad53-T358D mutation severely affects the kinase activity and causes accumulation of the S129- phosphorylated isoform of histone H2A, even during an unperturbed cell cycle, thus indicating the accumulation of spontaneous DNA breaks. We further found that the protein level of Sml1, which is the physiological inhibitor of ribonucleotide reductase, remains high during DNA replication in rad53-T358D cells, suggesting that an inadequate pool of dNTPs in checkpoint defective cells causes the accumulation of spontaneous DNA breaks.


Cell Cycle. 2007 Nov 16;7(4)

Fiorani S, Mimun G, Caleca L, Piccini D, Pellicioli A.
FIRC Institute of Molecular Oncology Foundation, Via Adamello 16, 20139 Milano, Italy.