Docking onto chromatin via the Saccharomyces cerevisiae Rad9 Tudor domain

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 .

The Dot1 Histone Methyltransferase and the Rad9 Checkpoint Adaptor Contribute to Cohesin-Dependent Double-Strand Break Repair by Sister Chromatid Recombination in Saccharomyces cerevisiae

“…In addition, it has been reported that Dot1-mediated histone H3K79 methylation is required for the recruitment of the Rad9 checkpoint adaptor protein to an irreparable HO-induced DSB at the chromosomal MAT locus (Wysocki et al 2005). Moreover, the formation of IR-induced Rad9 foci in G2 cells is impaired in the absence of Dot1 (Toh et al 2006;Grenon et al 2007), and the formation of MMS-induced Rad9 foci in G1 cells is also compromised in the dot1 mutant (supporting information, Figure S1). Therefore, it was possible that the SCR defect of dot1 results from failures to effectively recruit Rad9.…”

“…The only known direct target of Dot1 is H3K79 (van Leeuwen et al 2002), whose methylation contributes to Rad53 activation by promoting Rad9 recruitment to sites of damage ( Figure S1) (Giannattasio et al 2005;Wysocki et al 2005;Grenon et al 2007). As the Scc1 cohesin subunit undergoes DNA damage-induced phosphorylation, which is partially dependent on the Rad53 checkpoint kinase (Sidorova and Breeden 2003) and the rad53 mutant is partially defective in cohesin recruitment to a DSB (Unal et al 2004), it is possible that the effect of Dot1 and Rad9 in SCR is exerted by Rad53 activation, which in turn would promote cohesin loading.…”

Keep moving and stay in a good shape to find your homologous recombination partner

“…Notably, in both yeast and mammals, the checkpoint protein Rad9 53BP1 is constitutively bound to chromatin through the interaction between its Tudor domain and H3K79me (van Leeuwen et al 2002;Huyen et al 2004;Giannattasio et al 2005;Wysocki et al 2005;Grenon et al 2007). This interaction is further strengthened around DSB sites through interaction of its BRCT domain with DSB-induced γH2A (Javaheri et al 2006;Hammet et al 2007).…”

DNA Damage Leaves its Mark on Chromatin

“…18 While some modifications like H2AX phosphorylation (gH2AX) are induced by DNA damage, 19 others are normally cell-cycle regulated but persist after damage, as recently shown for H3K56 acetylation in yeast. 20 Finally, constitutive modifications can also provide specific binding sites for DNA damage response proteins, as described for methylation of H4K20 in fission yeast, 21 H3K79 in budding yeast 22,23 and both H3K79 and H4K20 in higher eukaryotes, 24,25 where determination of the relevant methylation site is still a matter of debate. While our knowledge of histone modifications involved in the damage response is steadily increasing, the role of ATP-dependent nucleosome remodeling in this process remains more elusive, especially in higher eukaryotes.…”