A truncated DNA-damage-signaling response is activated after DSB formation in the G1 phase of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the DNA damage response (DDR) is activated by the spatio-temporal colocalization of Mec1-Ddc2 kinase and the 9-1-1 clamp. In the absence of direct means to monitor Mec1 kinase activation in vivo, activation of the checkpoint kinase Rad53 has been taken as a proxy for DDR activation. Here, we identify serine 378 of the Rad55 recombination protein as a direct target site of Mec1. Rad55-S378 phosphorylation leads to an electrophoretic mobility shift of the protein and acts as a sentinel for Mec1 activation in vivo. A single double-stranded break (DSB) in G1-arrested cells causes phosphorylation of Rad55-S378, indicating activation of Mec1 kinase. However, Rad53 kinase is not detectably activated under these conditions. This response required Mec1-Ddc2 and loading of the 9-1-1 clamp by Rad24-RFC, but not Rad9 or Mrc1. In addition to Rad55–S378, two additional direct Mec1 kinase targets are phosphorylated, the middle subunit of the ssDNA-binding protein RPA, RPA2 and histone H2A (H2AX). These data suggest the existence of a truncated signaling pathway in response to a single DSB in G1-arrested cells that activates Mec1 without eliciting a full DDR involving the entire signaling pathway including the effector kinases

The DNA damage checkpoint pathways exert multiple controls on the efficiency and outcome of the repair of a double-stranded DNA gap

A DNA gap repair assay was used to determine the effect of mutations in the DNA damage checkpoint system on the efficiency and outcome (crossover/non-crossover) of recombinational DNA repair. In Saccharomyces cerevisiae gap repair is largely achieved by homologous recombination. As a result the plasmid either integrates into the chromosome (indicative of a crossover outcome) or remains extrachromosomal (indicative of a non-crossover outcome). Deletion mutants of the MEC1 and RAD53 checkpoint kinase genes exhibited a 5-fold decrease in gap repair efficiency, showing that 80% of the gap repair events depended on functional DNA damage checkpoints. Epistasis analysis suggests that the DNA damage checkpoints affect gap repair by modulating Rad51 protein-mediated homologous recombination. While in wild-type cells only ∼25% of the gap repair events were associated with a crossover outcome, Mec1-deficient cells exhibited a >80% crossover association. Also mutations in the effector kinases Rad53, Chk1 and Dun1 were found to affect crossover association of DNA gap repair to various degrees. The data suggest that the DNA damage checkpoints are important for the optimal functioning of recombinational DNA repair with multiple terminal targets to modulate the efficiency and outcome of homologous recombination

Direct Kinase-to-Kinase Signaling Mediated by the FHA Phosphoprotein Recognition Domain of the Dun1 DNA Damage Checkpoint Kinase

The serine-threonine kinase Dun1 contains a forkhead-associated (FHA) domain and functions in the DNA damage checkpoint pathway of Saccharomyces cerevisiae. It belongs to the Chk2 family of checkpoint kinases, which includes S. cerevisiae Rad53 and Mek1, Schizosaccharomyces pombe Cds1, and human Chk2. Dun1 is required for DNA damage-induced transcription of certain target genes, transient G(2)/M arrest after DNA damage, and DNA damage-induced phosphorylation of the DNA repair protein Rad55. Here we report that the FHA phosphoprotein recognition domain of Dun1 is required for direct phosphorylation of Dun1 by Rad53 kinase in vitro and in vivo. trans phosphorylation by Rad53 does not require the Dun1 kinase activity and is likely to involve only a transient interaction between the two kinases. The checkpoint functions of Dun1 kinase in DNA damage-induced transcription, G(2)/M cell cycle arrest, and Rad55 phosphorylation are severely compromised in an FHA domain mutant of Dun1. As a consequence, the Dun1 FHA domain mutant displays enhanced sensitivity to genotoxic stress induced by UV, methyl methanesulfonate, and the replication inhibitor hydroxyurea. We show that the Dun1 FHA domain is critical for direct kinase-to-kinase signaling from Rad53 to Dun1 in the DNA damage checkpoint pathway

A truncated DNA-damage-signaling response is activated after DSB formation in the G1 phase of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the DNA damage response (DDR) is activated by the spatio-temporal colocalization of Mec1-Ddc2 kinase and the 9-1-1 clamp. In the absence of direct means to monitor Mec1 kinase activation in vivo, activation of the checkpoint kinase Rad53 has been taken as a proxy for DDR activation. Here, we identify serine 378 of the Rad55 recombination protein as a direct target site of Mec1. Rad55-S378 phosphorylation leads to an electrophoretic mobility shift of the protein and acts as a sentinel for Mec1 activation in vivo. A single double-stranded break (DSB) in G1-arrested cells causes phosphorylation of Rad55-S378, indicating activation of Mec1 kinase. However, Rad53 kinase is not detectably activated under these conditions. This response required Mec1-Ddc2 and loading of the 9-1-1 clamp by Rad24-RFC, but not Rad9 or Mrc1. In addition to Rad55-S378, two additional direct Mec1 kinase targets are phosphorylated, the middle subunit of the ssDNA-binding protein RPA, RPA2 and histone H2A (H2AX). These data suggest the existence of a truncated signaling pathway in response to a single DSB in G1-arrested cells that activates Mec1 without eliciting a full DDR involving the entire signaling pathway including the effector kinases

Phosphorylation of Rad55 on Serines 2, 8, and 14 Is Required for Efficient Homologous Recombination in the Recovery of Stalled Replication Forks

DNA damage checkpoints coordinate the cellular response to genotoxic stress and arrest the cell cycle in response to DNA damage and replication fork stalling. Homologous recombination is a ubiquitous pathway for the repair of DNA double-stranded breaks and other checkpoint-inducing lesions. Moreover, homologous recombination is involved in postreplicative tolerance of DNA damage and the recovery of DNA replication after replication fork stalling. Here, we show that the phosphorylation on serines 2, 8, and 14 (S2,8,14) of the Rad55 protein is specifically required for survival as well as for normal growth under genome-wide genotoxic stress. Rad55 is a Rad51 paralog in Saccharomyces cerevisiae and functions in the assembly of the Rad51 filament, a central intermediate in recombinational DNA repair. Phosphorylation-defective rad55-S2,8,14A mutants display a very slow traversal of S phase under DNA-damaging conditions, which is likely due to the slower recovery of stalled replication forks or the slower repair of replication-associated DNA damage. These results suggest that Rad55-S2,8,14 phosphorylation activates recombinational repair, allowing for faster recovery after genotoxic stress

Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors

The myelodysplastic syndromes (MDSs) are collections of heterogeneous hematologic diseases characterized by refractory cytopenias as a result of ineffective hematopoiesis. Development of effective treatments has been impeded by limited insights into any unifying pathogenic pathways. We provide evidence that the p38 MAP kinase is constitutively activated or phosphorylated in MDS bone marrows. Such activation is uniformly observed in varied morphologic subtypes of low-risk MDS and correlates with enhanced apoptosis observed in MDS hematopoietic progenitors. Most importantly, pharmacologic inhibition of p38α by a novel small molecule inhibitor, SCIO-469, decreases apoptosis in MDS CD34+ progenitors and leads to dose-dependant increases in erythroid and myeloid colony formation. Down-regulation of the dominant p38α isoform by siRNA also leads to enhancement of hematopoiesis in MDS bone marrow progenitors in vitro. These data implicate p38 MAPK in the pathobiology of ineffective hematopoiesis in lowrisk MDS and provide a strong rationale for clinical investigation of SCIO-469 in MDS