Muriel Grenon

Checkpoint activation in response to double-strand breaks requires the Mre11/Rad50/Xrs2 complex.

Studies of human Nijmegen breakage syndrome (NBS) cells have led to the proposal that the Mre11/Rad50/ NBS1 complex, which is involved in the repair of DNA double-strand breaks (DSBs), might also function in activating the DNA damage checkpoint pathways after DSBs occur. We have studied the role of the homologous budding yeast complex, Mre11/Rad50/Xrs2, in checkpoint activation in response to DSB-inducing agents. Here we show that this complex is required for phosphorylation and activation of the Rad53 and Chk1 checkpoint kinases specifically in response to DSBs. Consistent with defective Rad53 activation, we observed defective cell-cycle delays after induction of DSBs in the absence of Mre11. Furthermore, after γ-irradiation phosphorylation of Rad9, which is an early event in checkpoint activation, is also dependent on Mre11. All three components of the Mre11/Rad50/Xrs2 complex are required for activation of Rad53, however, the Ku80, Rad51 or Rad52 proteins, which are also involved in DSB repair, are not. Thus, the integrity of the Mre11/Rad50/Xrs2 complex is specifically required for checkpoint activation after the formation of DSBs.

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.

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 G1 phase and its role in DNA repair is restricted to G2. Copyright

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.

Topoisomerase III Acts Upstream of Rad53p in the S-Phase DNA Damage Checkpoint

ABSTRACT Deletion of the Saccharomyces cerevisiae TOP3gene, encoding Top3p, leads to a slow-growth phenotype characterized by an accumulation of cells with a late S/G2content of DNA (S. Gangloff, J. P. McDonald, C. Bendixen, L. Arthur, and R. Rothstein, Mol. Cell. Biol. 14:8391–8398, 1994). We have investigated the function of TOP3 during cell cycle progression and the molecular basis for the cell cycle delay seen in top3Δ strains. We show that top3Δ mutants exhibit a RAD24-dependent delay in the G2 phase, suggesting a possible role for Top3p in the resolution of abnormal DNA structures or DNA damage arising during S phase. Consistent with this notion,top3Δ strains are sensitive to killing by a variety of DNA-damaging agents, including UV light and the alkylating agent methyl methanesulfonate, and are partially defective in the intra-S-phase checkpoint that slows the rate of S-phase progression following exposure to DNA-damaging agents. This S-phase checkpoint defect is associated with a defect in phosphorylation of Rad53p, indicating that, in the absence of Top3p, the efficiency of sensing the existence of DNA damage or signaling to the Rad53 kinase is impaired. Consistent with a role for Top3p specifically during S phase, top3Δ mutants are sensitive to the replication inhibitor hydroxyurea, expression of the TOP3 mRNA is activated in late G1 phase, and DNA damage checkpoints operating outside of S phase are unaffected by deletion of TOP3. All of these phenotypic consequences of loss of Top3p function are at least partially suppressed by deletion of SGS1, the yeast homologue of the human Blooms and Werners syndrome genes. These data implicate Top3p and, by inference, Sgs1p in an S-phase-specific role in the cellular response to DNA damage. A model proposing a role for these proteins in S phase is presented.

Transcriptional response of Candida parapsilosis following exposure to farnesol

ABSTRACT 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.

p56chk1 protein kinase is required for the DNA replication checkpoint at 37°C in fission yeast

Fission yeast p56chk1 kinase is known to be involved in the DNA damage checkpoint but not to be required for cell cycle arrest following exposure to the DNA replication inhibitor hydroxyurea (HU). For this reason, p56chk1 is considered not to be necessary for the DNA replication checkpoint which acts through the inhibitory phosphorylation of p34cdc2 kinase activity. In a search for Schizosaccharomyces pombe mutants that abolish the S phase cell cycle arrest of a thermosensitive DNA polymerase δ strain at 37°C, we isolated two chk1 alleles. These alleles are proficient for the DNA damage checkpoint, but induce mitotic catastrophe in several S phase thermosensitive mutants. We show that the mitotic catastrophe correlates with a decreased level of tyrosine phosphorylation of p34cdc2. In addition, we found that the deletion of chk1 and the chk1 alleles abolish the cell cycle arrest and induce mitotic catastrophe in cells exposed to HU, if the cells are grown at 37°C. These findings suggest that chk1 is important for the maintenance of the DNA replication checkpoint in S phase thermosensitive mutants and that the p56chk1 kinase must possess a novel function that prevents premature activation of p34cdc2 kinase under conditions of impaired DNA replication at 37°C.

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.

The MRN complex

Also known as… The Mre11 complex or MRX in yeast.What is MRN? A complex of three proteins — Mre11, Rad50 and Nbs1 (also known as Nibrin or p95). It is essential for the viability of vertebrate, but not yeast, cells. The MRN complex is engaged in DNA metabolic events involving DNA double-strand ends. Orthologues of human Rad50 and Mre11 have been identified in all taxonomic kingdoms whereas Nbs1 seems to be unique to eukaryotic cells as no orthologues have been identified in prokaryotes or archaebacteria. The well-characterized yeast homologue of MRN, MRX, contains the Mre11, Rad50 and Xrs2 proteins, the later showing weak homology to human Nbs1.Why is it essential for vertebrate cell viability? The genetic material of all eukaryotic cells is constantly exposed to both endogenous and exogenous DNA-damaging agents. Even a single double-stand break (DSB) can be lethal. Left unrepaired, DSBs can lead to chromosome instability, rearrangements, gene mutations and cancer. It is therefore extremely important for the cell to be able to sense the break, signal this damage and effect the appropriate biological responses as soon as possible. The MRN complex functions in both sensing and signaling of DSBs. It also has roles in both major DSB repair pathways — homologous recombination (HR) and non-homologous end joining (NHEJ). The MRN complex is also required for cell-cycle checkpoint signaling after DSB in all phases of the cell cycle. Additionally, it plays an important role in processing DNA structures that arise during normal S phase, is involved in preventing DNA re-replication and is essential for telomere maintenance.How do Mre11, Rad50 and Nbs1 contribute to MRN function? The three members of the complex have distinct roles within the intact MRN complex. Mre11 interacts with both Rad50 and Nbs1, which do not directly contact each other (Figure 1Figure 1). Rad50 has two globular domains linked by a long coiled-coil region forming extended arms. At the end of each arm a hook domain allows Rad50 molecules to dimerise and tether DNA ends together (Figure 1Figure 1). Mre11 is responsible for DNA binding and also has both exo- and endonuclease activities, which have been characterized in vitro, and an ability to unwind DNA locally. Finally, Nbs1, which has no known enzymatic activities, is responsible for the rapid re-localization of the complex into large focal structures, as well as for most of the interactions with other DSB-signaling and DNA-repair proteins. Its binding partners include ATM, γH2AX and MDC1. The carboxy-terminal region of Nbs1 has also been reported to regulate irradiation-induced apoptosis (Figure 1Figure 1).Figure 1Domain structure of the MRN components.Domains responsible for interactions within the complex are shown in yellow. CXXC hook, zinc hook; FHA, Forkhead associated domain; BRCT, BRCA1 carboxyl terminus domain. Note that the PAR domain of Mre11 localized between the two DNA binding motifs is not shown.View Large Image | View Hi-Res Image | Download PowerPoint SlideIs there a connection between MRN and cancer? Yes. Mre11, Rad50 and Nbs1 are known tumor suppressors. Loss of function of any of these proteins results in genome instability, the principal feature of cancer cells. Defective MRN function has been linked to many types of cancer, including breast, ovarian, colorectal, gastric and prostate cancers, as well as leukemia and melanoma. Hypomorphic mutations in any of the human genes for MRN result in cancer predisposing genome-instability syndromes: mutations in the Mre11 and Nbs1 genes cause ataxia telangiectasia-like disorder (ATLD) and Nijmegen breakage syndrome (NBS), respectively. Mutation of RAD50 has been described recently but so far there is no syndrome associated with it. The symptoms of MRN syndromes are largely overlapping and include immunodeficiency and mental deficiency.How does MRN respond to DNA damage? Immediately after the DSB induction, MRN re-localizes to the sites of damage (Figure 2Figure 2A). Initial recruitment is probably via its end-binding activity but, subsequently, excess MRN is recruited to the vicinity of DNA damage in large focal structures centered around DSBs. Focal accumulation is regulated by interaction with γH2AX, the DNA-damage-specific phosphoform of histone H2AX. During initial recruitment MRN is thought to bind and secure the DNA ends together via zinc hooks at the ends of the long flexible Rad50 arms, with the Mre11 molecules binding to DSBs (Figure 2Figure 2B). Upon DSB binding the Rad50 arms undergo structural change, becoming rigid and parallel and bridging both DNA ends using the zinc hook (Figure 2Figure 2B,D). Secured this way, the initial steps of DSB processing can take place. In addition, a signaling cascade is activated with the initial step being activation of the ATM kinase (Figure 2Figure 2D). Inactive ATM dimers are believed to be recruited to Nbs1 at DSBs, resulting in autophosphorylation and dissociation as active ATM monomers. The ATM kinase then phosphorylates many DNA-damage response proteins, including Nbs1 itself, resulting in the induction of the DNA-damage signaling cascade. Many DNA-damage response proteins are also recruited to the sites of damage and contribute to DSB processing and repair (Figure 2Figure 2C). ATM-dependent signaling contributes to efficient DSB repair, transcription and apoptosis, as well as regulating transient cell-cycle arrest, which is believed to allow sufficient time for DNA repair before the key cell-cycle transition.Figure 2A model presenting the initial steps of the DNA-damage response.(A) DSB induction. The MRN complex binds to DSBs and tethers DNA ends. PARP1 attaches ADP-ribose units to chromatin-bound proteins. Note that it is possible that DNA is secured by more than one copy of the complex at each DNA end. (B) The local concentration of MRN complex is increased by the interaction of Nbs1 with γH2AX and Mre11 binding to ADP-ribose units on chromatin-bound proteins. Note that the structure of the complex bound to DNA ends (rigid arms) differs from the unbound form (more flexible arms). At the same time, active ATM monomers phosphorylate downstream targets including Nbs1 and the signaling cascade is activated

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.