Phosphorylation of the budding yeast 9-1-1 complex is required for Dpb11 function in the full activation of the UV-induced DNA damage checkpoint.
ABSTRACT Following genotoxic insults, eukaryotic cells trigger a signal transduction cascade known as the DNA damage checkpoint response, which involves the loading onto DNA of an apical kinase and several downstream factors. Chromatin modifications play an important role in recruiting checkpoint proteins. In budding yeast, methylated H3-K79 is bound by the checkpoint factor Rad9. Loss of Dot1 prevents H3-K79 methylation, leading to a checkpoint defect in the G(1) phase of the cell cycle and to a reduction of checkpoint activation in mitosis, suggesting that another pathway contributes to Rad9 recruitment in M phase. We found that the replication factor Dpb11 is the keystone of this second pathway. dot1Delta dpb11-1 mutant cells are sensitive to UV or Zeocin treatment and cannot activate Rad53 if irradiated in M phase. Our data suggest that Dpb11 is held in proximity to damaged DNA through an interaction with the phosphorylated 9-1-1 complex, leading to Mec1-dependent phosphorylation of Rad9. Dpb11 is also phosphorylated after DNA damage, and this modification is lost in a nonphosphorylatable ddc1-T602A mutant. Finally, we show that, in vivo, Dpb11 cooperates with Dot1 in promoting Rad9 phosphorylation but also contributes to the full activation of Mec1 kinase.
Article: The transcription factor C/EBP-beta and its role in ovarian function; evidence for direct involvement in the ovulatory process.[show abstract] [hide abstract]
ABSTRACT: Gonadotropins are responsible for maturation of the ovarian follicle and the oocyte. Ovulation is the ultimate step in this process and involves disintegration of the follicular wall and subsequent release of an oocyte into the oviduct. These events are triggered by a surge of luteinizing hormone (LH). Genes expressed in the ovary, that respond to LH, are likely to be involved in the biochemical pathways that regulate ovulation. The transcription factor C/EBP-beta is induced promptly in the ovary, as a response to an ovulatory dose of gonadotropins. We used an ex vivo perfusion system to demonstrate that a specific reduction in ovarian C/EBP-beta expression inhibits ovulation. In such ovaries the oocytes appeared to be entrapped within the follicle. We have found a correlation between the expression level of the activating isoform of C/EBP-beta and the number of oocytes ovulated in response to gonadotropins. Since a reduction in C/EBP-beta expression does not affect the level of the ovulatory mediator prostaglandin endoperoxide synthase-2 (PGS-2), these findings support the view of C/EBP-beta as an important factor in the ovulatory process and highlight a C/EBP-beta-dependent and PGS-2-independent pathway that takes part in regulation of ovulation.The EMBO Journal 10/1997; 16(17):5273-9. · 9.20 Impact Factor
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ABSTRACT: An integrated cellular response to DNA damage is essential for the maintenance of genome integrity. Recently, post-translational modifications to histone proteins have been implicated in DNA damage responses involving the Rad9 family of checkpoint proteins. In budding yeast, methylation of histone H3 on lysine 79 (H3-K79me) has been shown to be required for efficient checkpoint signalling and Rad9 localization on chromatin. Here, we have used a rad9 Tudor mutant allele and cells mutated for Dot1, the H3-K79 methylase, to analyse the epistatic relationship between RAD9 and DOT1 genes regarding the DNA damage resistance and checkpoint activation pathways. Our results show that RAD9 is epistatic to DOT1 and suggest that it acts downstream of the Dot1 methylase in the damage resistance and checkpoint response. We have also found that the Tudor domain of Rad9 is necessary for in vitro binding to H3-K79me as well as Rad9 focal accumulation in response to DNA damage in vivo. In summary, our study demonstrates that the interaction between Rad9, via its Tudor domain, and methylated H3-K79 is required at two different steps of the DNA damage response, an early step corresponding to checkpoint activation, and a late step corresponding to DNA repair. The study further shows that the function of this interaction is cell cycle-regulated; the role in checkpoint activation is restricted to the G(1) phase and its role in DNA repair is restricted to G(2).Yeast 03/2007; 24(2):105-19. · 1.89 Impact Factor