A DNA damage response pathway controlled by Tel1 and the Mre11 complex.

We define a DNA damage checkpoint pathway in S. cerevisiae governed by the ATM homolog Tel1 and the Mre11 complex. In mitotic cells, the Tel1-Mre11 complex pathway triggers Rad53 activation and its interaction with Rad9, whereas in meiosis it acts via Rad9 and the Rad53 paralog Mre4/Mek1. Activation of the Tel1-Mre11 complex pathway checkpoint functions appears to depend upon the Mre11 complex as a damage sensor and, at least in meiotic cells, to depend on unprocessed DNA double-strand breaks (DSBs). The DSB repair functions of the Mre11 complex are enhanced by the pathway, suggesting that the complex both initiates and is regulated by the Tel1-dependent DSB signal. These findings demonstrate that the diverse functions of the Mre11 complex in the cellular DNA damage response are conserved in mammals and yeast.     

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 gamma-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.     

The Mre11 complex is required for ATM activation and the G2/M checkpoint

The maintenance of genome integrity requires a rapid and specific response to many types of DNA damage. The conserved and related PI3-like protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate signal transduction pathways in response to genomic insults, such as DNA double-strand breaks (DSBs). It is unclear which proteins recognize DSBs and activate these pathways, but the Mre11/Rad50/NBS1 complex has been suggested to act as a damage sensor. Here we show that infection with an adenovirus lacking the E4 region also induces a cellular DNA damage response, with activation of ATM and ATR. Wild-type virus blocks this signaling through degradation of the Mre11 complex by the viral E1b55K/E4orf6 proteins. Using these viral proteins, we show that the Mre11 complex is required for both ATM activation and the ATM-dependent G(2)/M checkpoint in response to DSBs. These results demonstrate that the Mre11 complex can function as a damage sensor upstream of ATM/ATR signaling in mammalian cells.     

Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins

DNA repair is an essential process for preserving genome integrity in all organisms. In eukaryotes, recombinational repair is choreographed by multiprotein complexes that are organized into centers (foci). Here, we analyze the cellular response to DNA double-strand breaks (DSBs) and replication stress in Saccharomyces cerevisiae. The Mre11 nuclease and the ATM-related Tel1 kinase are the first proteins detected at DSBs. Next, the Rfa1 single-strand DNA binding protein relocalizes to the break and recruits other key checkpoint proteins. Later and only in S and G2 phase, the homologous recombination machinery assembles at the site. Unlike the response to DSBs, Mre11 and recombination proteins are not recruited to hydroxyurea-stalled replication forks unless the forks collapse. The cellular response to DSBs and DNA replication stress is likely directed by the Mre11 complex detecting and processing DNA ends in conjunction with Sae2 and by RP-A recognizing single-stranded DNA and recruiting additional checkpoint and repair proteins.     

PIKK-dependent phosphorylation of Mre11 induces MRN complex inactivation by disassembly from chromatin

The role of Mre11 phosphorylation in the cellular response to DNA double-strand breaks (DSBs) is not well understood. Here, we show that phosphorylation of Mre11 at SQ/TQ motifs by PIKKs (PI3 Kinase-related Kinases) induces MRN (Mre11–Rad50–Nbs1) complex dissociation from chromatin by reducing Mre11 affinity for DNA. Whereas phosphorylation of Mre11 at these residues is not required for DSB-induced ATM (Ataxia-Telangiectasia mutated) activation, abrogation of Mre11 dephosphorylation impairs ATM signaling. Our study provides a functional characterization of the DNA damage-induced Mre11 phosphorylation, and suggests that MRN inactivation participates in the down-regulation of damage signaling during checkpoint recovery following DSB repair.     

Requirement of the Mre11 complex and exonuclease 1 for activation of the Mec1 signaling pathway

The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate DNA damage checkpoint pathways. In budding yeast, ATM and ATR homologs are encoded by TEL1 and MEC1, respectively. The Mre11 complex consists of two highly related proteins, Mre11 and Rad50, and a third protein, Xrs2 in budding yeast or Nbs1 in mammals. The Mre11 complex controls the ATM/Tel1 signaling pathway in response to double-strand break (DSB) induction. We show here that the Mre11 complex functions together with exonuclease 1 (Exo1) in activation of the Mec1 signaling pathway after DNA damage and replication block. Mec1 controls the checkpoint responses following UV irradiation as well as DSB induction. Correspondingly, the Mre11 complex and Exo1 play an overlapping role in activation of DSB- and UV-induced checkpoints. The Mre11 complex and Exo1 collaborate in producing long single-stranded DNA (ssDNA) tails at DSB ends and promote Mec1 association with the DSBs. The Ddc1-Mec3-Rad17 complex associates with sites of DNA damage and modulates the Mec1 signaling pathway. However, Ddc1 association with DSBs does not require the function of the Mre11 complex and Exo1. Mec1 controls checkpoint responses to stalled DNA replication as well. Accordingly, the Mre11 complex and Exo1 contribute to activation of the replication checkpoint pathway. Our results provide a model in which the Mre11 complex and Exo1 cooperate in generating long ssDNA tracts and thereby facilitate Mec1 association with sites of DNA damage or replication block.     

Distinct roles of ATR and DNA‐PKcs in triggering DNA damage responses in ATM‐deficient cells

The cellular response to DNA double‐strand breaks involves direct activation of ataxia telangiectasia mutated (ATM) and indirect activation of ataxia telangiectasia and Rad3 related (ATR) in an ATM/Mre11/cell‐cycle‐dependent manner. Here, we report that the crucial checkpoint signalling proteins—p53, structural maintainance of chromosomes 1 (SMC1), p53 binding protein 1 (53BP1), checkpoint kinase (Chk)1 and Chk2—are phosphorylated rapidly by ATR in an ATM/Mre11/cell‐cycle‐independent manner, albeit at low levels. We observed the sequential recruitment of replication protein A (RPA) and ATR to the sites of DNA damage in ATM‐deficient cells, which provides a mechanistic basis for the observed phosphorylations. The recruitment of ATR and consequent phosphorylations do not require Mre11 but are dependent on Exo1. We show that these low levels of phosphorylation are biologically important, as ATM‐deficient cells enforce an early G2/M checkpoint that is ATR‐dependent. ATR is also essential for the late G2 accumulation that is peculiar to irradiated ATM‐deficient cells. Interestingly, phosphorylation of KRAB associated protein 1 (KAP‐1), a protein involved in chromatin remodelling, is mediated by DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) in a spatio‐temporal manner in addition to ATM. We posit that ATM substrates involved in cell‐cycle checkpoint signalling can be minimally phosphorylated independently by ATR, while a small subset of proteins involved in chromatin remodelling are phosphorylated by DNA‐PKcs in addition to ATM.     

The MRE11 GAR motif regulates DNA double-strand break processing and ATR activation

The MRE11/RAD50/NBS1 complex is the primary sensor rapidly recruited to DNA double-strand breaks (DSBs). MRE11 is known to be arginine methylated by PRMT1 within its glycine-arginine-rich (GAR) motif. In this study, we report a mouse knock-in allele of Mre11 that substitutes the arginines with lysines in the GAR motif and generates the MRE11(RK) protein devoid of methylated arginines. The Mre11(RK/RK) mice were hypersensitive to γ-irradiation (IR) and the cells from these mice displayed cell cycle checkpoint defects and chromosome instability. Moreover, the Mre11(RK/RK) MEFs exhibited ATR/CHK1 signaling defects and impairment in the recruitment of RPA and RAD51 to the damaged sites. The M(RK)RN complex formed and localized to the sites of DNA damage and normally activated the ATM pathway in response to IR. The M(RK)RN complex exhibited exonuclease and DNA-binding defects in vitro responsible for the impaired DNA end resection and ATR activation observed in vivo in response to IR. Our findings provide genetic evidence for the critical role of the MRE11 GAR motif in DSB repair, and demonstrate a mechanistic link between post-translational modifications at the MRE11 GAR motif and DSB processing, as well as the ATR/CHK1 checkpoint signaling.     

Defective Mre11-dependent activation of Chk2 by ataxia telangiectasia mutated in colorectal carcinoma cells in response to replication-dependent DNA double strand breaks

The Mre11.Rad50.Nbs1 (MRN) complex binds DNA double strand breaks to repair DNA and activate checkpoints. We report MRN deficiency in three of seven colon carcinoma cell lines of the NCI Anticancer Drug Screen. To study the involvement of MRN in replication-mediated DNA double strand breaks, we examined checkpoint responses to camptothecin, which induces replication-mediated DNA double strand breaks after replication forks collide with topoisomerase I cleavage complexes. MRN-deficient cells were deficient for Chk2 activation, whereas Chk1 activation was independent of MRN. Chk2 activation was ataxia telangiectasia mutated (ATM)-dependent and associated with phosphorylation of Mre11 and Nbs1. Mre11 complementation in MRN-deficient HCT116 cells restored Chk2 activation as well as Rad50 and Nbs1 levels. Conversely, Mre11 down-regulation by small interference RNA (siRNA) in HT29 cells inhibited Chk2 activation and down-regulated Nbs1 and Rad50. Proteasome inhibition also restored Rad50 and Nbs1 levels in HCT116 cells suggesting that Mre11 stabilizes Rad50 and Nbs1. Chk2 activation was also defective in three of four MRN-proficient colorectal cell lines because of low Chk2 levels. Thus, six of seven colon carcinoma cell lines from the NCI Anticancer Drug Screen are functionally Chk2-deficient in response to replication-mediated DNA double strand breaks. We propose that Mre11 stabilizes Nbs1 and Rad50 and that MRN activates Chk2 downstream from ATM in response to replication-mediated DNA double strand breaks. Chk2 deficiency in HCT116 is associated with defective S-phase checkpoint, prolonged G2 arrest, and hypersensitivity to camptothecin. The high frequency of MRN and Chk2 deficiencies may contribute to genomic instability and therapeutic response to camptothecins in colorectal cancers.     

Promotion of Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes.

S. cerevisiae RAD50, MRE11, and XRS2 genes are required for telomere maintenance, cell cycle checkpoint signaling, meiotic recombination, and the efficient repair of DNA double-strand breaks (DSB)s by homologous recombination and nonhomologous end-joining (NHEJ). Here, we demonstrate that the complex formed by Rad50, Mre11, and Xrs2 proteins promotes intermolecular DNA joining by DNA ligase IV (Dnl4) and its associated protein Lif1. Our results show that the Rad50/Mre11/Xrs2 complex juxtaposes linear DNA molecules via their ends to form oligomers and interacts directly with Dnl4/Lif1. We also demonstrate that Rad50/Mre11/Xrs2-mediated intermolecular DNA joining is further stimulated by Hdf1/Hdf2, the yeast homolog of the mammalian Ku70/Ku80 heterodimer. These studies reveal specific functional interplay among the Hdf1/Hdf2, Rad50/Mre11/Xrs2, and Dnl4/Lif1 complexes in NHEJ.     

Localization of checkpoint and repair proteins in eukaryotes

In eukaryotes, the cellular response to DNA damage depends on the type of DNA structure being recognized by the checkpoint and repair machinery. DNA ends and single-stranded DNA are hallmarks of double-strand breaks and replication stress. These two structures are recognized by distinct sets of proteins, which are reorganized into a focal assembly at the lesion. Moreover, the composition of these foci is coordinated with cell cycle progression, reflecting the favoring of end-joining in the G1 phase and homologous recombination in S and G2. The assembly of proteins at sites of DNA damage is largely controlled by a network of protein-protein interactions, with the Mre11 complex initiating assembly at DNA ends and replication protein A directing recruitment to single-stranded DNA. This review summarizes current knowledge on the cellular organization of DSB repair and checkpoint proteins focusing on budding yeast and mammalian cells.     

Cdc13 telomere capping decreases Mec1 association but does not affect Tel1 association with DNA ends

Chromosome ends, known as telomeres, have to be distinguished from DNA breaks that activate DNA damage checkpoint. Two large protein kinases, ataxia-teleangiectasia mutated (ATM) and ATM-Rad3-related (ATR), control not only checkpoint activation but also telomere length. In budding yeast, Mec1 and Tel1 correspond to ATR and ATM, respectively. Here, we show that Cdc13-dependent telomere capping attenuates Mec1 association with DNA ends. The telomeric TG repeat sequence inhibits DNA degradation and decreases Mec1 accumulation at the DNA end. The TG-mediated degradation block requires binding of multiple Cdc13 proteins. The Mre11-Rad50-Xrs2 complex and Exo1 contribute to DNA degradation at DNA ends. Although the TG sequence impedes Exo1 association with DNA ends, it allows Mre11 association. Moreover, the TG sequence does not affect Tel1 association with the DNA end. Our results suggest that the Cdc13 telomere cap coordinates Mec1 and Tel1 accumulation rather than simply covering the DNA ends at telomeres.     

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.     

The cellular response to DNA double-strand breaks: defining the sensors and mediators

The induction of DNA double-strand breaks (DSBs) culminates in the activation of cell cycle checkpoint responses and DNA repair machinery. The mechanism of DSB detection remains unclear although many candidate sensor proteins have been identified through cytologic, biochemical and genetic studies. In light of recent advances in our understanding of the cellular response to DSBs, we have proposed criteria for defining sensor proteins. We discuss the possible role of the Mre11 complex as a primary damage sensor and the complex relationship between DNA damage sensors, transducers and mediators.     

Mre11 dimers coordinate DNA end bridging and nuclease processing in double-strand-break repair

Mre11 forms the core of the multifunctional Mre11-Rad50-Nbs1 (MRN) complex that detects DNA double-strand breaks (DSBs), activates the ATM checkpoint kinase, and initiates homologous recombination (HR) repair of DSBs. To define the roles of Mre11 in both DNA bridging and nucleolytic processing during initiation of DSB repair, we combined small-angle X-ray scattering (SAXS) and crystal structures of Pyrococcus furiosus Mre11 dimers bound to DNA with mutational analyses of fission yeast Mre11. The Mre11 dimer adopts a four-lobed U-shaped structure that is critical for proper MRN complex assembly and for binding and aligning DNA ends. Further, mutations blocking Mre11 endonuclease activity impair cell survival after DSB induction without compromising MRN complex assembly or Mre11-dependant recruitment of Ctp1, an HR factor, to DSBs. These results show how Mre11 dimerization and nuclease activities initiate repair of DSBs and collapsed replication forks, as well as provide a molecular foundation for understanding cancer-causing Mre11 mutations in ataxia telangiectasia-like disorder (ATLD).     

Double functions for the Mre11 complex during DNA double-strand break repair and replication

Defining the factors that lead to genomic instability is one of the most important fields in cancer biology. DNA damage can arise from exogenous sources or as a result of normal cellular metabolism. Regardless of the cause, when damaged DNA is not properly repaired the genome acquires mutation(s). Under normal circumstances, to prevent such chromosome instability the cell activates the checkpoint response, which inhibits cell cycle progression until DNA repair is complete. The Mre11 complex is formed by three components: Mre11, Rad50, and Nbs1/Xrs2 and is involved in the signaling pathways that lead to both checkpoint activation and DNA repair. In response to DNA damage two functions of the complex will be discussed, one involves its role in initiating kinase activation and the second involves its ability to tether and link DNA strands. This review will highlight the functions of the Mre11 complex during the process of DNA double strand break recognition and repair, and during the process of replication. Understanding how the Mre11 complex is working at the molecular level is important for understanding why disruptions in components of the complex lead to genomic instability and cancer predisposition syndromes in humans.     

The Mre11 complex and ATM: a two-way functional interaction in recognising and signaling DNA double strand breaks

Mutations in components of the Mre11/Rad50/Nbs1 complex give rise to genetic disorders characterized by neurological abnormalities, radiosensitivity, cell cycle checkpoint defects, genomic instability and cancer predisposition. Evidence exists that this complex associates with chromatin during DNA replication and acts as a sensor of double strand breaks (dsbs) in DNA after exposure to radiation. A series of recent reports provides additional support that the complex senses breaks in DNA and relays this information to ATM, mutated in ataxia-telangiectasia (A-T), which in turn activates pathways for cell cycle checkpoint activation. Paradoxically members of the Mre11 complex are also downstream of ATM in these pathways. Here, Lavin attempts to make sense of this sensing mechanism with reference to a series of recent reports on the topic.     

Mre11 ATLD17/18 mutation retains Tel1/ATM activity but blocks DNA double-strand break repair

The Mre11 complex (Mre11-Rad50-Nbs1 or MRN) binds double-strand breaks where it interacts with CtIP/Ctp1/Sae2 and ATM/Tel1 to preserve genome stability through its functions in homology-directed repair, checkpoint signaling and telomere maintenance. Here, we combine biochemical, structural and in vivo functional studies to uncover key properties of Mre11-W243R, a mutation identified in two pediatric cancer patients with enhanced ataxia telangiectasia-like disorder. Purified human Mre11-W243R retains nuclease and DNA binding activities in vitro. X-ray crystallography of Pyrococcus furiosus Mre11 indicates that an analogous mutation leaves the overall Mre11 three-dimensional structure and nuclease sites intact but disorders surface loops expected to regulate DNA and Rad50 interactions. The equivalent W248R allele in fission yeast allows Mre11 to form an MRN complex that efficiently binds double-strand breaks, activates Tel1/ATM and maintains telomeres; yet, it causes hypersensitivity to ionizing radiation and collapsed replication forks, increased Rad52 foci, defective Chk1 signaling and meiotic failure. W248R differs from other ataxia telangiectasia-like disorder analog alleles by the reduced stability of its interaction with Rad50 in cell lysates. Collective results suggest a separation-of-function mutation that disturbs interactions amongst the MRN subunits and Ctp1 required for DNA end processing in vivo but maintains interactions sufficient for Tel1/ATM checkpoint and telomere maintenance functions.     

Regulation of Mre11/Rad50 by Nbs1: effects on nucleotide-dependent DNA binding and association with ataxia-telangiectasia-like disorder mutant complexes

The Mre11/Rad50 complex is a critical component of the cellular response to DNA double-strand breaks, in organisms ranging from archaebacteria to humans. In mammalian cells, Mre11/Rad50 (M/R) associates with a third component, Nbs1, that regulates its activities and is targeted by signaling pathways that initiate DNA damage-induced checkpoint responses. Mutations in the genes that encode Nbs1 and Mre11 are responsible for the human radiation sensitivity disorders Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD), respectively, which are characterized by defective checkpoint responses and high levels of chromosomal abnormalities. Here we demonstrate nucleotide-dependent DNA binding by the human M/R complex that requires the Nbs1 protein and is specific for double-strand DNA duplexes. Efficient DNA binding is only observed with non-hydrolyzable analogs of ATP, suggesting that ATP hydrolysis normally effects DNA release. The alleles of MRE11 associated with ATLD and the C-terminal Nbs1 polypeptide associated with NBS were expressed with the other components and found to form triple complexes except in the case of ATLD 3/4, which exhibits variability in Nbs1 association. The ATLD 1/2, ATLD 3/4, and p70 M/R/N complexes exhibit nucleotide-dependent DNA binding and exonuclease activity equivalent to the wild-type enzyme, although the ATLD complexes both show reduced activity in endonuclease assays. Sedimentation equilibrium analysis of the recombinant human complexes indicates that Mre11 is a stable dimer, Mre11 and Nbs1 form a 1:1 complex, and both M/R and M/R/N form large multimeric assemblies of approximately 1.2 MDa. Models of M/R/N stoichiometry in light of this and previous data are discussed.