HISTONE-DEPENDENT AND HISTONE-INDEPENDENT PATHWAYS FOR RAD9 CHROMATIN RECRUITMENT AND CHECKPOINT ACTIVATION

Maintenance of genome stability is critical to cell survival and normal cell growth. Indeed, genome instability is a hallmark of most human cancers. To ensure the accurate transmission of genetic information to the offspring, eukaryotic cells evolved a complex network of surveillance and DNA repair mechanism, which allow the faithful transmission of genetic information throughout generations. These surveillance systems, called DNA damage checkpoints, are signal transduction cascades where the DNA damage signal is transmitted, through the action of protein kinases, to the cell cycle machinery, resulting in the temporary arrest of cell proliferation at the G1/s or at the G2/M transitions, or in the slowing down of DNA replication. Moreover checkpoint activation frequently brings about also changes in the transcritptional programme of the cell and modification in the DNA repair factors, resulting in a more efficient removal of the lesion. Since the basic checkpoint response has been shown to be conserved, from yeast to human cells, many details of this mechanism have been outlined by genetic and biochemical means in budding and fission yeasts, thanks to their genetic versatility and experimental tractability. The current model predicts that activation of the first checkpoint kinase in the cascade is not due to the DNA damage itself, but it requires recognition and initial processing of the lesion by some nucleases generating long 3’ ssDNA tails. The ssDNA regions are rapidly bound by the RPA complex, generating a structure that is responsible for the recruitment and activation of the apical kinase complex Mec1-Ddc2 and for the loading onto DNA of the 9-1-1 complex. Once activated, Mec1 phosphorylates different targets, among which Ddc2, the Ddc1 component of the 9-1-1 complex, histone H2A. Another Mec1 target is adaptor Rad9. Phosphorylation of Rad9, followed by its oligomerization, allows the recruitment and activation of the effector checkpoint kinase Rad53. This is a key step in the signal transduction cascade; it can be easily visualized as a hyperphosphorylated slower-mobility form by Western blotting and it is generally used as a marker to monitor checkpoint activation. Recent work demonstrated that histone H2B-K123 ubiquitylation, carried out by Rad6-Bre1, and histone H3 methylation on lysine 79 (H3-K79), performed by Dot1, contribute to Rad9 recruitment to chromatin. Infact, it has been demonstrated that Rad9 physically interacts with methylated H3-K79 thanks to its Tudor domain. Impairment of this recruitment-pathway prevents Rad9 and Rad53 phosphorylation in G1-arrested cells and abolishes the G1-S arrest following DNA damage. Surprisingly in M-arrested cells, deletion of Dot1 or mutation of the Rad9 Tudor domain does not completely abrogate the checkpoint function and Rad53 phosphorylation after genotoxic treatment. This evidence suggests the existence of a second pathway, partially redundant with the histone dependent branch, that, in M phase, provides an alternative way for Rad9 to be recruited in the proximity of the lesion and to be phosphorylated. We found that the replication factor Dpb11 is the keystone of this second pathway. Our data suggest that Dpb11 is held in proximity to damaged DNA through an interaction with phosphorylated 9-1-1, specifically within its Ddc1 subunit. Once recruited in the proximity of the lesion, it performs a double role: it contributes in the full activation of the apical checkpoint kinase Mec1 and it cooperates with Dot1 in the recruitment of Rad9. In particular, we discocvered that Dpb11 physically interacts with Rad9. This interaction depends upon CDK-dependent phosphorylation of Rad9 on the Ser11 residue, at the N-terminus of the protein. We also provide evidence that the Dpb11-dependent branch of Rad9 recruitment is necessary and sufficient for checkpoint activation when the histone-dependent pathway is impaired, and it allows Rad53 phosphorylation, despite undetectable Rad9 binding on the chromatin, suggesting that Rad9 complexed with Dpb11 is not tighly linked to chromatin.