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  • Vincristine clinical The Rev protein is a crucial regulator


    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 Vincristine clinical [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. In summary, our studies provide insight into how a chromatin modification, namely Dot1-dependent H3K79 methylation, regulates the tolerance to alkylating damage by TLS through modulation of Rad53 activity. TLS constitutes one important aspect of the coordinated global cellular response to DNA damage because of the ability to bypass lesions that impede replication progression, thus preventing fork collapse and eventual formation of DNA breaks potentially leading to chromosomal rearrangements. However, given the error-prone nature of TLS, this process must be kept under strict control to avoid excessive mutagenesis, which can also have deleterious consequences. Therefore, an appropriate balance between error-prone and error-free processes to face DNA damage is essential to avoid genomic instability, which is directly linked to cancer development. Our studies in yeast reveal that the conserved Rad53 checkpoint kinase contributes to finely tune this balance at least by regulating the levels of chromatin-bound Rev1. Recent studies using a mouse model point to the influence of correct Rev1 levels in reducing the incidence of carcinogen-induced lung cancer [73], highlighting the importance of these mechanisms for the maintenance of genomic stability.
    Conflict of interest
    Acknowledgements We thank M. Foiani, T. Hishida, R. Rothstein, M.A. Osley and G.C. Walker for providing plasmids and/or strains. We are indebted to F. van Leeuwen for sharing reagents and results prior to publication. We are especially grateful to R. Freire for raising the anti-Dot1 antibody and to F. Prado and J.A. Tercero for comments on the manuscript. DO and AGS were supported by predoctoral fellowships from CSIC and MEC (Spain), respectively. Research in our labs is supported by grants from Ministry of Science and Innovation of Spain (BFU2009-06938 to AB and BFU2009-07159 to PSS) and a grant from Fundación Ramón Areces to PSS.