The SAD1/RAD53 protein kinase controls multiple checkpoints and DNA damage-induced transcription in yeast.

Abstract

Inhibition of DNA synthesis prevents mitotic entry through the action of the S-phase checkpoint. We have isolated S-phase arrest-defective {sad) mutants that show lethality in the presence of the DNA synthesis inhibitor hydroxyurea (HU). Several of these mutants show phenotypes consistent with inappropriate mitotic entry in the presence of unreplicated DNA, indicating a defect in the S-phase checkpoint, sadl mutants are additionally defective for the G^ and Gj DNA damage checkpoints, and for DNA damage-induced transcription of RNR2 and RNR3. The transcriptional response to DNA damage requires activation of the Dunl protein kinase. Activation of Dunl in response to replication blocks or DNA damage is blocked in sadl mutants. The HU sensitivity of sadl mutants is suppressed by mutations in CKSl, a subunit of the p34^"*^^* kinase, further establishing a link between cell cycle progression and lethality, sadl mutants are allelic to rad53, a radiation-sensitive mutant. SADl encodes an essential protein kinase. The observation that SADl controls three distinct checkpoints suggests a common mechanism for cell cycle arrest at these points. Together, these observations implicate protein phosphorylation in the cellular response to DNA damage and replication blocks. A successful eukaryotic cell division requires that cer tain cellular processes occur in a defined order and are coupled so that the initiation of one event is dependent on the completion of another. In most cell types, entry into mitosis is dependent on the completion of DNA synthesis. Cells blocked for DNA replication arrest in S phase and delay mitotic initiation. Cell cycle arrest in response to S-phase inhibition is attributable to the pres ence of a feedback control or checkpoint mechanism (for reviews on cell cycle checkpoints, see Hartwell and

Mitotic DNA damage and replication checkpoints in yeast

“…This distinction is made on the basis of the additive phenotypes of rad9 sc Δ in combination with deletions of members of the RAD24 sc epistasis group [18,46•,47••]. The other two genes required for the metaphase DNA damage checkpoint, MEC1 sc and RAD53 sc , are also required for the S/M checkpoint [12,48]. They also differ in that they are essential for viability but this role is independent of their mitotic checkpoint functions (SJ Elledge, personal communication).…”

“…While, Tel1 sc is not required for checkpoints in S. cerevisiae, it can substitute for Mec1 sc in a number of situations [50,51•,52]. Rad53 sc is also a protein kinase and it contains two forkhead associated (FHA) domains, one on either end of the protein [48,53], which may be involved in binding regulatory partners. Dun1 sc is a Rad53 sc homolog that is also involved in checkpoints [54].…”

Regulation of the antioncogenic Chk2 kinase by the oncogenic Wip1 phosphatase

“…[1][2][3][4][5][6][7][8] Upon DNA damage, Chk2 is activated by phosphorylation of threonine68 (Thr68) by ATM (ataxiatelangiectasia mutated). [9][10][11][12][13] Activated Chk2 then phosphorylates its downstream effectors, including the tumour suppressors p53, BRCA1, PML, E2F-1, and Cdc25 phosphatases, [14][15][16][17][18][19] thereby regulating cellular responses following DNA damage.…”

Global Gene Expression Responses of Fission Yeast to Ionizing Radiation

“…In S. cerevisiae, RNR2 and RNR3, which encode the small and large subunits of ribonucleotide reductase, respectively (Elledge and Davis, 1987;Hurd et al, 1987), are transcriptionally induced in a checkpoint-dependent manner: After activation of the Mec1-Rad53-Dun1 checkpoint pathway, Crt1 (a transcriptional repressor) is hyperphosphorylated. This leads to its promoter-dissociation and the subsequent derepession of the RNR2 gene in response to DNA damage (Zhou and Elledge, 1993;Allen et al, 1994;Huang et al, 1998). Microarray analysis has recently identified a Mec1-dependent gene expression signature in response to DNAdamaging agents that includes a large set of genes responsive to diverse environmental stresses (Gasch et al, 2001).…”