Toh, G. W. et al. Histone H2A phosphorylation and H3 methylation are required for a novel Rad9 DSB repair function following checkpoint activation. DNA Repair (Amst.) 5, 693-703
ABSTRACT In budding yeast, the Rad9 protein is an important player in the maintenance of genomic integrity and has a well-characterised role in DNA damage checkpoint activation. Recently, roles for different post-translational histone modifications in the DNA damage response, including H2A serine 129 phosphorylation and H3 lysine 79 methylation, have also been demonstrated. Here, we show that Rad9 recruitment to foci and bulk chromatin occurs specifically after ionising radiation treatment in G2 cells. This stable recruitment correlates with late stages of double strand break (DSB) repair and, surprisingly, it is the hypophosphorylated form of Rad9 that is retained on chromatin rather than the hyperphosphorylated, checkpoint-associated, form. Stable Rad9 accumulation in foci requires the Mec1 kinase and two independently regulated histone modifications, H2A phosphorylation and Dot1-dependent H3 methylation. In addition, Rad9 is selectively recruited to a subset of Rad52 repair foci. These results, together with the observation that rad9Delta cells are defective in repair of IR breaks in G2, strongly indicate a novel post checkpoint activation role for Rad9 in promoting efficient repair of DNA DSBs by homologous recombination.
- SourceAvailable from: Jeff Thompson
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- "It has been previously shown by our group and others that histone H3K79 methylation is important with respect to UV damage response. Yeast cells lacking Dot1, the H3K79 methyltransferase, are hypersensitive to UV relative to wild-type cells (11), display defects in the G1/S DNA damage checkpoint (14–16), and have reduced nucleotide excision repair (20,21). Histone H2B K123 ubiquitylation is also important for the response to UV, as cells lacking this modification similarly possess G1/S checkpoint deficiencies (14–16). "
ABSTRACT: Histone post-translational modifications have been shown to contribute to DNA damage repair. Prior studies have suggested that specific H3K79 methylation states play distinct roles in the response to UV-induced DNA damage. To evaluate these observations, we examined the effect of altered H3K79 methylation patterns on UV-induced G1/S checkpoint response and sister chromatid exchange (SCE). We found that the di- and trimethylated states both contribute to activation of the G1/S checkpoint to varying degrees, depending on the synchronization method, although methylation is not required for checkpoint in response to high levels of UV damage. In contrast, UV-induced SCE is largely a product of the trimethylated state, which influences the usage of gene conversion versus popout mechanisms. Regulation of H3K79 methylation by H2BK123 ubiquitylation is important for both checkpoint function and SCE. H3K79 methylation is not required for the repair of double-stranded breaks caused by transient HO endonuclease expression, but does play a modest role in survival from continuous exposure. The overall results provide evidence for the participation of H3K79 methylation in UV-induced recombination repair and checkpoint activation, and further indicate that the di- and trimethylation states play distinct roles in these DNA damage response pathways.Nucleic Acids Research 04/2014; 42(10). DOI:10.1093/nar/gku242 · 9.11 Impact Factor
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- "The yeast Dot1 deletion mutant decreases and/or retards the phosphorylation of the 53BP1 ortholog Rad9 and the Chk2 homolog Rad53 kinase to activate the G1- and intra-S checkpoint following DNA damage induced by ultraviolet (UV), X-ray, and ionizing radiation (IR).94,96,97 Dot1 is required to recruit Rad9 to DSBs, although whether the Tudor domain of Rad9 directly interacts with methylated H3K79 remains a matter of debate.96,98-100 Furthermore, loss of Rad9 binding to histone H3 increases DSB resection, which occurs during homologous recombination, thereby generating single-stranded DNA (ssDNA).101 "
ABSTRACT: Dot1/DOT1L catalyzes the methylation of histone H3 lysine 79 (H3K79), which regulates diverse cellular processes, such as development, reprogramming, differentiation, and proliferation. In regards to these processes, studies of Dot1/DOT1L-dependent H3K79 methylation have mainly focused on the transcriptional regulation of specific genes. Although the gene transcription mediated by Dot1/DOT1L during the cell cycle is not fully understood, H3K79 methylation plays a critical role in the progression of G 1 phase, S phase, mitosis, and meiosis. This modification may contribute to the chromatin structure that controls gene expression, replication initiation, DNA damage response, microtubule reorganization, chromosome segregation, and heterochromatin formation. Overall, Dot1/DOT1L is required to maintain genomic and chromosomal stability. This review summarizes the several functions of Dot1/DOT1L and highlights its role in cell cycle regulation.Cell cycle (Georgetown, Tex.) 02/2014; 13(5). DOI:10.4161/cc.28104 · 5.01 Impact Factor
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- "This could be readout for loss of proper checkpoint function, however accumulating evidence suggests that Rad9 may play multiple roles in the DNA damage response. For instance, Rad9 has been implicated in nucleotide excision repair of UV-damaged DNA , , for suppression of mutagenic post-replicative repair during MMS induced damage , and a post checkpoint activation role for Rad9 in promoting efficient repair of DSBs imposed by irradiation has also been suggested . Thus, it seems we are far from understanding the complexity of the cellular functions that Rad9 may be engaged in to act as a caretaker gene. "
ABSTRACT: The conserved family of RecQ DNA helicases consists of caretaker tumour suppressors, that defend genome integrity by acting on several pathways of DNA repair that maintain genome stability. In budding yeast, Sgs1 is the sole RecQ helicase and it has been implicated in checkpoint responses, replisome stability and dissolution of double Holliday junctions during homologous recombination. In this study we investigate a possible genetic interaction between SGS1 and RAD9 in the cellular response to methyl methane sulphonate (MMS) induced damage and compare this with the genetic interaction between SGS1 and RAD24. The Rad9 protein, an adaptor for effector kinase activation, plays well-characterized roles in the DNA damage checkpoint response, whereas Rad24 is characterized as a sensor protein also in the DNA damage checkpoint response. Here we unveil novel insights into the cellular response to MMS-induced damage. Specifically, we show a strong synergistic functionality between SGS1 and RAD9 for recovery from MMS induced damage and for suppression of gross chromosomal rearrangements, which is not the case for SGS1 and RAD24. Intriguingly, it is a Rad53 independent function of Rad9, which becomes crucial for genome maintenance in the absence of Sgs1. Despite this, our dissection of the MMS checkpoint response reveals parallel, but unequal pathways for Rad53 activation and highlights significant differences between MMS- and hydroxyurea (HU)-induced checkpoint responses with relation to the requirement of the Sgs1 interacting partner Topoisomerase III (Top3). Thus, whereas earlier studies have documented a Top3-independent role of Sgs1 for an HU-induced checkpoint response, we show here that upon MMS treatment, Sgs1 and Top3 together define a minor but parallel pathway to that of Rad9.PLoS ONE 11/2013; 8(11):e81015. DOI:10.1371/journal.pone.0081015 · 3.23 Impact Factor
Geraldine Wei-Ling Toh