The role of Dot in multiple nuclear

The role of Dot1 in multiple nuclear processes, such as transcriptional silencing, meiotic checkpoint, DNA damage checkpoint or DSB repair, relies on the regulated binding of various key factors to specific chromosomal regions [25], [26], [27], [33]. In principle, the impact of Dot1 (i.e. H3K79 methylation) in MMS resistance and the higher number of MMS-induced chromosome-associated Rev1 foci in the dot1Δ mutant could emanate from a direct effect of a peculiar chromatin structure dictated by the H3K79 methylation status modulating the recruitment of the TLS machinery. However, our results support an alternative possibility implying that the effect of Dot1 in DNA damage tolerance is exerted indirectly through the regulation of the Rad53 checkpoint kinase. The fact that a rad53-HA mutant, characterized by reduced levels of the kinase, substantially phenocopies dot1Δ in the response to chronic MMS exposure ([34], [40]; this work) suggested a possible relationship between Dot1 and Rad53 in the regulation of tolerance to alkylating damage. Supporting this possibility, we find that the MMS resistance of both dot1Δ and rad53-HA depends on ubiquitylation of PCNA at K164, which is a crucial regulator of the TLS mechanism of DNA damage tolerance. Moreover, we show here that the increased MMS resistance of the dot1Δ mutant depends on Rad53. The Rev1 protein is a crucial regulator of TLS activity because of its structural function [10]; therefore, we focused on Rev1 to investigate how Dot1/Rad53 function impinges on TLS-dependent mutagenic bypass of MMS-induced lesions. In particular, we examined Rev1 localization to chromatin by immunofluorescence of nuclear spreads. We found that Rev1 foci are present in most nuclei even in the absence of MMS damage, suggesting that there is a constitutive localization of Rev1 to chromosomes. Similar results have been reported for 4NQO-treated ski or skii [61]. Since PCNA ubiquitylation is triggered by DNA damage [11], these observations imply that the basal formation of Rev1 foci does not depend on the interaction with ubiquitylated PCNA. Consistent with this possibility, we detect Rev1 foci in the ubiquitylation-deficient pol30-K164R mutant (Supplementary Fig. 1). In fact, studies of mouse and yeast Rev1 suggest that the BRCT domain of Rev1 is required for its constitutive recruitment to foci, whereas the ubiquitin-binding motifs specifically drive Rev1 to damaged replication forks [60], [69], [70]. Moreover, in DT40 chicken cells, Rev1 maintains progression of replication forks upon DNA damage independently of PCNA ubiquitylation [71]. Strikingly, although most nuclei maintain Rev1 signal, we observe a decrease in the number of Rev1 foci per nucleus in MMS-treated wild-type cells. Since mutagenic TLS is induced by alkylating damage [34], this reduced number of Rev1 foci (or a significant fraction of them) must by actively engaged in TLS. In contrast, the number of chromatin-bound Rev1 foci remains elevated in the dot1Δ or rad53-HA mutants, providing the opportunity for more TLS-dependent mutagenic events once DNA damage-induced ubiquitylation of PCNA occurs. We propose that full activation of the Rad53 checkpoint kinase, which depends on Dot1, somehow restrains TLS activity by preventing promiscuous formation of Rev1 foci associated with chromosomes. Rev1 undergoes Mec1-dependent phosphorylation, which promotes Polζ activity only in NER-deficient cells [61], [72]. Phosphorylation of Rev1 also requires the checkpoint clamp ‘9-1-1′ and the clamp loader Rad24; however, it is independent of Rad53 [72]. Therefore, it is unlikely that this posttranslational modification of Rev1 controls the formation of TLS-active Rev1 foci. Perhaps, Rad53 acts on other regulators of TLS that mediate Rev1 chromosomal binding or stability. Future studies will be aimed to unveil these mechanisms.