ING3 is required for ATM signaling and DNA repair in response to DNA double strand breaks.

Inhibitor of Growth 3 (ING3) is a candidate tumor suppressor gene whose expression is lost in tumors such as hepatocellular carcinoma, head and neck squamous cell carcinoma and melanoma. In the present study, we show that ING3-depleted human cells and yeast cells deleted for its ortholog YNG2 are sensitive to DNA damage suggesting a conserved role in response to such stress. In human cells, ING3 is recruited to DNA double strand breaks and is required for ATM activation. Remarkably, in response to doxorubicin, ATM activation is dependent on ING3 but not on TIP60, whose recruitment to DNA breaks also depends on ING3. These events lead to ATM-mediated phosphorylation of NBS1 and the subsequent recruitment of RNF8, RNF168, 53BP1, and BRCA1, which are major mediators of the DNA damage response. Accordingly, upon genotoxic stress, DNA repair by non-homologous end joining (NHEJ) or homologous recombination (HR) were impaired in absence of ING3. Finally, immunoglobulin class switch recombination (CSR), a physiological mechanism requiring NHEJ repair, was impaired in the absence of ING3. Since deregulation of DNA double strand break repair is associated with genomic instability, we propose a novel function of ING3 as a caretaker tumor suppressor involved in the DNA damage signaling and repair.

Researchers: share your passion for science!

The promotion of the public understanding of science has many positive impacts on society, including expanding the reach of science to a broader range of individuals and having a favourable impact on the economy. It also results in many benefits for researchers involved, including the development of their communication skills and improvement in the quality of their research. Despite increased awareness of the importance of public engagement (PE), the involvement of researchers has only slightly increased in the last 10 years. Time constraints, lack of opportunity and lack of funding are the main barriers preventing their participation. We propose that joining an existing PE programme can be a good way for scientists to overcome these barriers. We list specific examples of established activities that are easy for researchers to get involved in, allowing them to share their enthusiasm for science.

Site-specific phosphorylation of the DNA damage response mediator rad9 by cyclin-dependent kinases regulates activation of checkpoint kinase 1.

The mediators of the DNA damage response (DDR) are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK) complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR-specific protein kinases.

Eukaryotic DNA damage checkpoint activation in response to double-strand breaks.

Double-strand breaks (DSBs) are the most detrimental form of DNA damage. Failure to repair these cytotoxic lesions can result in genome rearrangements conducive to the development of many diseases, including cancer. The DNA damage response (DDR) ensures the rapid detection and repair of DSBs in order to maintain genome integrity. Central to the DDR are the DNA damage checkpoints. When activated by DNA damage, these sophisticated surveillance mechanisms induce transient cell cycle arrests, allowing sufficient time for DNA repair. Since the term "checkpoint" was coined over 20 years ago, our understanding of the molecular mechanisms governing the DNA damage checkpoint has advanced significantly. These pathways are highly conserved from yeast to humans. Thus, significant findings in yeast may be extrapolated to vertebrates, greatly facilitating the molecular dissection of these complex regulatory networks. This review focuses on the cellular response to DSBs in Saccharomyces cerevisiae, providing a comprehensive overview of how these signalling pathways function to orchestrate the cellular response to DNA damage and preserve genome stability in eukaryotic cells.

Regulation of the DNA damage response and gene expression by the Dot1L histone methyltransferase and the 53Bp1 tumour suppressor.

BACKGROUND: Dot1L, a histone methyltransferase that targets histone H3 lysine 79 (H3K79), has been implicated in gene regulation and the DNA damage response although its functions in these processes remain poorly defined. METHODOLOGY/PRINCIPAL FINDINGS: Using the chicken DT40 model system, we generated cells in which the Dot1L gene is disrupted to examine the function and focal recruitment of the 53Bp1 DNA damage response protein. Detailed kinetic and dose response assays demonstrate that, despite the absence of H3K79 methylation demonstrated by mass spectrometry, 53Bp1 focal recruitment is not compromised in these cells. We also describe, for the first time, the phenotypes of a cell line lacking both Dot1L and 53Bp1. Dot1L⁻/⁻ and wild type cells are equally resistant to ionising radiation, whereas 53Bp1⁻/⁻/Dot1L⁻/⁻ cells display a striking DNA damage resistance phenotype. Dot1L and 53Bp1 also affect the expression of many genes. Loss of Dot1L activity dramatically alters the mRNA levels of over 1200 genes involved in diverse biological functions. These results, combined with the previously reported list of differentially expressed genes in mouse ES cells knocked down for Dot1L, demonstrates surprising cell type and species conservation of Dot1L-dependent gene expression. In 53Bp1⁻/⁻ cells, over 300 genes, many with functions in immune responses and apoptosis, were differentially expressed. To date, this is the first global analysis of gene expression in a 53Bp1-deficient cell line. CONCLUSIONS/SIGNIFICANCE: Taken together, our results uncover a negative role for Dot1L and H3K79 methylation in the DNA damage response in the absence of 53Bp1. They also enlighten the roles of Dot1L and 53Bp1 in gene expression and the control of DNA double-strand repair pathways in the context of chromatin.

Dynamics of Rad9 chromatin binding and checkpoint function are mediated by its dimerization and are cell cycle-regulated by CDK1 activity.

Saccharomyces cerevisiae Rad9 is required for an effective DNA damage response throughout the cell cycle. Assembly of Rad9 on chromatin after DNA damage is promoted by histone modifications that create docking sites for Rad9 recruitment, allowing checkpoint activation. Rad53 phosphorylation is also dependent upon BRCT-directed Rad9 oligomerization; however, the crosstalk between these molecular determinants and their functional significance are poorly understood. Here we report that, in the G1 and M phases of the cell cycle, both constitutive and DNA damage-dependent Rad9 chromatin association require its BRCT domains. In G1 cells, GST or FKBP dimerization motifs can substitute to the BRCT domains for Rad9 chromatin binding and checkpoint function. Conversely, forced Rad9 dimerization in M phase fails to promote its recruitment onto DNA, although it supports Rad9 checkpoint function. In fact, a parallel pathway, independent on histone modifications and governed by CDK1 activity, allows checkpoint activation in the absence of Rad9 chromatin binding. CDK1-dependent phosphorylation of Rad9 on Ser11 leads to specific interaction with Dpb11, allowing Rad53 activation and bypassing the requirement for the histone branch.

MRN and the race to the break.

In all living cells, DNA is constantly threatened by both endogenous and exogenous agents. In order to protect genetic information, all cells have developed a sophisticated network of proteins, which constantly monitor genomic integrity. This network, termed the DNA damage response, senses and signals the presence of DNA damage to effect numerous biological responses, including DNA repair, transient cell cycle arrests ("checkpoints") and apoptosis. The MRN complex (MRX in yeast), composed of Mre11, Rad50 and Nbs1 (Xrs2), is a key component of the immediate early response to DNA damage, involved in a cross-talk between the repair and checkpoint machinery. Using its ability to bind DNA ends, it is ideally placed to sense and signal the presence of double strand breaks and plays an important role in DNA repair and cellular survival. Here, we summarise recent observation on MRN structure, function, regulation and emerging mechanisms by which the MRN nano-machinery protects genomic integrity. Finally, we discuss the biological significance of the unique MRN structure and summarise the emerging sequence of early events of the response to double strand breaks orchestrated by the MRN complex.

53BP1: function and mechanisms of focal recruitment.

53BP1 (p53-binding protein 1) is classified as a mediator/adaptor of the DNA-damage response, and is recruited to nuclear structures termed foci following genotoxic insult. In the present paper, we review the functions of 53BP1 in DNA-damage checkpoint activation and DNA repair, and the mechanisms of its recruitment and activation following DNA damage. We focus in particular on the role of covalent histone modifications in this process.

Transcriptional response of Candida parapsilosis following exposure to farnesol.

In Candida albicans, the quorum-sensing molecule farnesol inhibits the transition from yeast to hyphae but has no effect on cellular growth. We show that the addition of exogenous farnesol to cultures of Candida parapsilosis causes the cells to arrest, but not at a specific stage in the cell cycle. The cells are not susceptible to additional farnesol. However, the cells do eventually recover from arrest. Unlike in C. albicans, in C. parapsilosis sterols are localized to the tips of budding cells, and this polarization is disrupted by the addition of farnesol. We used the results of a genome sequence survey to design and manufacture partial genomic microarrays that were applied to determining the transcriptional response of C. parapsilosis to the presence of exogenous farnesol. In both C. albicans and C. parapsilosis, exposure to farnesol results in increased expression of the oxidoreductases GRP2 and ADH7 and altered expression of genes involved in sterol metabolism. There is no effect on expression of C. parapsilosis orthologs of genes involved in hyphal growth in C. albicans. Farnesol therefore differs significantly in its effects on C. parapsilosis and C. albicans.

Docking onto chromatin via the Saccharomyces cerevisiae Rad9 Tudor domain.

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).

Double-strand breaks trigger MRX- and Mec1-dependent, but Tel1-independent, checkpoint activation.

Together with the Tel1 PI3 kinase, the Mre11/Rad50/Xrs2 (MRX) complex is involved in checkpoint activation in response to double-strand breaks (DSBs), a function also conserved in human cells by Mre11/Rad50/Nbs1 acting with ATM. It has been proposed that the yeast Tel1/MRX pathway is activated in the presence of DSBs that cannot be resected. The Mec1 PI3 kinase, by contrast, would be involved in detecting breaks that can be processed. The significance of a Mec1/MRX DSB-activated DNA damage checkpoint has yet to be reported. To understand whether the MRX complex works specifically with Tel1 or Mec1, we investigated MRX function in checkpoint activation in response to endonuclease-induced DSBs in synchronized cells. We found that the expression of EcoRI activated the G1 and intra-S phase checkpoints in a MRX- and Mec1-dependent, but Tel1-independent manner. The pathways identified here are therefore different from the Tel1/MRX pathway that was previously reported. Thus, our results demonstrate that MRX can function in concert with both Mec1 and Tel1 PI3K-like kinases to trigger checkpoint activation in response to DSBs. Importantly, we also describe a novel MRX-independent checkpoint that is activated in late S-phase when cells replicate their DNA in the presence of DSBs. The existence of this novel mode of checkpoint activation explains why several previous studies had reported that mutations in the MRX complex did not abrogate DSB-induced checkpoint activation in asynchronous cells.

Multiple approaches to study S. cerevisiae Rad9, a prototypical checkpoint protein.

The Saccharomyces cerevisiae RAD9 checkpoint gene is the prototypical checkpoint gene and is required for efficient checkpoint regulation in late G1, S, and at the G2/M cell cycle transition following DNA damage. Rad9 is required for the activation of Rad53 after damage and has been proposed to have roles in lesion recognition as well as DNA repair and the maintenance of genome stability. Here we describe methodology suitable for the study of G1, intra-S, and G2/M checkpoints in budding yeast, the analysis of Rad9/Rad53 phospho-forms, the biochemical analysis of Rad9 and Rad53, the fractionation of soluble and chromatin associated proteins, including Rad9, and the live cell imaging of GFP tagged Rad9.

Histone H2A phosphorylation and H3 methylation are required for a novel Rad9 DSB repair function following checkpoint activation.

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.

The budding yeast Rad9 checkpoint complex: chaperone proteins are required for its function.

Rad9 functions in the DNA-damage checkpoint pathway of Saccharomyces cerevisiae. In whole-cell extracts, Rad9 is found in large, soluble complexes, which have functions in amplifying the checkpoint signal. The two main soluble forms of Rad9 complexes that are found in cells exposed to DNA-damaging treatments were purified to homogeneity. Both of these Rad9 complexes contain the Ssa1 and/or Ssa2 chaperone proteins, suggesting a function for these proteins in checkpoint regulation. Consistent with this possibility, genetic experiments indicate redundant functions for SSA1 and SSA2 in survival, G2/M-checkpoint regulation, and phosphorylation of both Rad9 and Rad53 after irradiation with ultraviolet light. Ssa1 and Ssa2 can now be considered as novel checkpoint proteins that are likely to be required for remodelling Rad9 complexes during checkpoint-pathway activation.

Fission yeast chk1 mutants show distinct responses to different types of DNA damaging treatments.

BACKGROUND: Chk1 kinase is activated by phosphorylation at serine-345 by Rad3 checkpoint kinase and is required for DNA damage checkpoint in late S and G2 phase of S. pombe cell cycle. We studied the ability of two chk1 mutants, chk1-1 and chk1-2, to undergo phosphorylation and to delay cell cycle progression in response to different types of DNA lesions. RESULTS: Both the Chk1-1 and Chk1-2 mutant proteins are phosphorylated to various extents when DNA is damaged in early G2 phase of cell cycle by either UV irradiation or gamma irradiation. However, chk1-2 mutant does not delay cell cycle progression in a dose dependent manner specifically upon gamma irradiations. This defect is not associated with an important loss of survival. Furthermore, both chk1 mutants survive to Camptothecin treatment despite undetectable Chk1-1 or Chk1-2 phosphorylated forms. We show that both mutant proteins are not phosphorylated in cds1 devoid cells treated with ribonucleotide reductase inhibitor hydroxyurea or when the replisome is affected by a thermosensitive mutation in DNA polymerase delta. This inability is associated with the loss of checkpoint function. We found that an increased level of Crb2/Rhp9 protein specifically complements the defect of the chk1-1 mutant allowing Chk1-1 phosphorylation upon treatment with hydroxyurea of dcds1 cells. CONCLUSIONS: Mutants chk1-1 and chk1-2 behave differently according to the type of lesion generated on DNA.