Dpb11 and the 9-1-1 Complex Act Redundantly in Promoting Checkpoint Activation after Replication Stress

Following DNA damage or replication stress, yeast cells phosphorylate and activate the Rad53 (hChk2) checkpoint protein, which is responsible for maintaining genome stability in these challenging conditions. The activation mechanism of the DNA damage checkpoint and of the replication checkpoint is different, but partially overlapping because most of checkpoint factors are shared between these pathways. For the activation of the two checkpoints the upstream kinase complex Mec1/Ddc2 (hATR/hATRIP) is required and both the PCNA-like complex and the replicative factor Dpb11 (hTopBP1) play a relevant role. We have previously shown that in UV damaged yeast cells, Dpb11 is required for the histone methylation-independent function of Rad9. Dpb11 has been also reported to be able to stimulate in vitro the Mec1 kinase activity. To better understand in vivo the function of Dpb11 in activating the apical kinase, we decided to study checkpoint activation after treatment with hydroxyurea (HU), an inhibitor of ribonucleotide reductase. In fact, HU induces Rad53 phosphorylation independently of the Rad9 adaptor protein allowing us to study the function of Dpb11 in the activation of Mec1 and its interplay with 9-1-1 complex (Ddc1-Mec3-Rad17). We show here that a ddc1Δdpb11-1 double mutant strain displays a Rad53 phosphorylation defect after HU treatment, similar to the one of a mec1-1 mutant. Moreover, the double mutant also lacks the phosphorylation of histone H2A. These observations suggest that Dpb11 and the PCNA-like complex act independently in promoting Mec1 activation. A similar phenotype can be observed in a dpb4Δddc1Δ strain carrying a deletion of a non-essential subunit of DNA polymerase ε, indicating that Dpb11 may be working together with Polε in this function