Gerald E. Dodson

Nbs1 Flexibly Tethers Ctp1 and Mre11-Rad50 to Coordinate DNA Double-Strand Break Processing and Repair

The Nijmegen breakage syndrome 1 (Nbs1) subunit of the Mre11-Rad50-Nbs1 (MRN) complex protects genome integrity by coordinating double-strand break (DSB) repair and checkpoint signaling through undefined interactions with ATM, MDC1, and Sae2/Ctp1/CtIP. Here, fission yeast and human Nbs1 structures defined by X-ray crystallography and small angle X-ray scattering (SAXS) reveal Nbs1 cardinal features: fused, extended, FHA-BRCT(1)-BRCT(2) domains flexibly linked to C-terminal Mre11- and ATM-binding motifs. Genetic, biochemical, and structural analyses of an Nbs1-Ctp1 complex show Nbs1 recruits phosphorylated Ctp1 to DSBs via binding of the Nbs1 FHA domain to a Ctp1 pThr-Asp motif. Nbs1 structures further identify an extensive FHA-BRCT interface, a bipartite MDC1-binding scaffold, an extended conformational switch, and the molecular consequences associated with cancer predisposing Nijmegen breakage syndrome mutations. Tethering of Ctp1 to a flexible Nbs1 arm suggests a mechanism for restricting DNA end processing and homologous recombination activities of Sae2/Ctp1/CtIP to the immediate vicinity of DSBs.

Ataxia-telangiectasia-mutated (ATM) is a T-antigen kinase that controls SV40 viral replication in vivo.

The structurally related ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) protein kinases fulfill overlapping yet non-redundant functions as key regulators of cellular DNA damage responses. We recently showed that ATM phosphorylates the cyclic AMP response element-binding protein, CREB, following exposure to ionizing radiation (IR) and other DNA-damaging stimuli. Here, we show that a phospho-specific antibody recognizing the major ATM phosphorylation site in CREB cross-reacts with SV40 large tumor antigen (LTag), a multifunctional oncoprotein required for replication of the SV40 minichromosome. The relevant IR-induced phosphorylation site in LTag recognized by phospho-CREB antibody was mapped to Ser-120. IR strongly induced the phosphorylation of Ser-120 in an ATM-dependent manner in mouse embryo fibroblasts. Infection of African green monkey CV1 cells with SV40 resulted in the activation of ATM and phosphorylation of LTag and endogenous ATM substrates. Infection-induced LTag phosphorylation correlated with the onset of DNA replication, was ATM-dependent, and peaked when viral DNA levels reached their maximum. SV40 replication in CV1 cells required an intact LTag Ser-120 phosphorylation site and was inhibited following transfection with ATM small interfering RNA suggesting that ATM is required for optimal SV40 replication in primate cells. Our findings uncover a direct link between ATM and SV40 LTag that may have implications for understanding the replication cycle of oncogenic polyoma viruses.

Identification of Carboxyl-terminal MCM3 Phosphorylation Sites Using Polyreactive Phosphospecific Antibodies

The functionally related ATM (ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) protein kinases are critical regulators of DNA damage responses in mammalian cells. ATM and ATR share highly overlapping substrate specificities and show a strong preference for the phosphorylation of Ser or Thr residues followed by Gln. In this report we used a polyreactive phosphospecific antibody (α-pDSQ) that recognizes a subset of phosphorylated Asp-Ser-Gln sequences to purify candidate ATM/ATR substrates. This led to the identification of phosphorylation sites in the carboxyl terminus of the minichromosome maintenance protein 3 (MCM3), a component of the hexameric MCM DNA helicase. We show that the α-DSQ antibody recognizes tandem DSQ phosphorylation sites (Ser-725 and Ser-732) in the carboxyl terminus of murine MCM3 (mMCM3) and that ATM phosphorylates both sites in vitro. ATM phosphorylated the carboxyl termini of mMCM3 and human MCM3 in vivo and the phosphorylated form of MCM3 retained association with the canonical MCM complex. Although DNA damage did not affect steady-state levels of chromatin-bound MCM3, the ATM-phosphorylated form of MCM3 was preferentially localized to the soluble, nucleoplasmic fraction. This finding suggests that the carboxyl terminus of chromatin-loaded MCM3 may be sequestered from ATM-dependent checkpoint signals. Finally, we show that ATM and ATR jointly contribute to UV light-induced MCM3 phosphorylation, but that ATM is the predominant UV-activated MCM3 kinase in vivo. The carboxyl-terminal ATM phosphorylation sites are conserved in vertebrate MCM3 orthologs suggesting that this motif may serve important regulatory functions in response to DNA damage. Our findings also suggest that DSQ motifs are common phosphoacceptor motifs for ATM family kinases.

DNA Replication Stress-induced Phosphorylation of Cyclic AMP Response Element-binding Protein Mediated by ATM

The DNA damage-response regulators ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) are structurally and functionally related protein kinases that exhibit nearly identical substrate specificities in vitro. Current paradigms hold that the relative contributions of ATM and ATR to nuclear substrate phosphorylation are dictated by the type of initiating DNA lesion; ATM-dependent substrate phosphorylation is principally activated by DNA double strand breaks, whereas ATR-dependent substrate phosphorylation is induced by UV light and other forms of DNA replication stress. In this report, we employed the cyclic AMP-response element-binding (CREB) protein to provide evidence for substrate discrimination by ATM and ATR in cellulo. ATM and ATR phosphorylate CREB in vitro, and CREB is phosphorylated on Ser-121 in intact cells in response to ionizing radiation (IR), UV light, and hydroxyurea. The UV light- and hydroxyurea-induced phosphorylation of CREB was delayed in comparison to the canonical ATR substrate CHK1, suggesting potentially different mechanisms of phosphorylation. UV light-induced CREB phosphorylation temporally correlated with ATM autophosphorylation on Ser-1981, and an ATM-specific small interfering RNA suppressed CREB phosphorylation in response to this stimulus. UV light-induced CREB phosphorylation was absent in ATM-deficient cells, confirming that ATM is required for CREB phosphorylation in UV irradiation-damaged cells. Interestingly, RNA interference-mediated suppression of ATR partially inhibited CREB phosphorylation in response to UV light, which correlated with reduced phosphorylation of ATM on Ser-1981. These findings suggest that ATM is the major genotoxin-induced CREB kinase in mammalian cells and that ATR lies upstream of ATM in a UV light-induced signaling pathway.

Mutations in String/CDC25 inhibit cell cycle re-entry and neurodegeneration in a Drosophila model of Ataxia telangiectasia

Mutations in ATM (Ataxia telangiectasia mutated) result in Ataxia telangiectasia (A-T), a disorder characterized by progressive neurodegeneration. Despite advances in understanding how ATM signals cell cycle arrest, DNA repair, and apoptosis in response to DNA damage, it remains unclear why loss of ATM causes degeneration of post-mitotic neurons and why the neurological phenotype of ATM-null individuals varies in severity. To address these issues, we generated a Drosophila model of A-T. RNAi knockdown of ATM in the eye caused progressive degeneration of adult neurons in the absence of exogenously induced DNA damage. Heterozygous mutations in select genes modified the neurodegeneration phenotype, suggesting that genetic background underlies variable neurodegeneration in A-T. The neuroprotective activity of ATM may be negatively regulated by deacetylation since mutations in a protein deacetylase gene, RPD3, suppressed neurodegeneration, and a human homolog of RPD3, histone deacetylase 2, bound ATM and abrogated ATM activation in cell culture. Moreover, knockdown of ATM in post-mitotic neurons caused cell cycle re-entry, and heterozygous mutations in the cell cycle activator gene String/CDC25 inhibited cell cycle re-entry and neurodegeneration. Thus, we hypothesize that ATM performs a cell cycle checkpoint function to protect post-mitotic neurons from degeneration and that cell cycle re-entry causes neurodegeneration in A-T.

Phosphorylation-regulated binding of Ctp1 to Nbs1 is critical for repair of DNA double-strand breaks

Repair of DNA double-strand breaks (DSBs) is critical for cell survival and for maintaining genome stability in eukaryotes. In Schizosaccharomyces pombe, the Mre11-Rad50-Nbs1 (MRN) complex and Ctp1 cooperate to perform the initial steps that process and repair these DNA lesions via homologous recombination (HR). While Ctp1 is recruited to DSBs in an MRN-dependent manner, the specific mechanism of this process remained unclear. We recently found that Ctp1 is phosphorylated on a domain rich in putative Casein kinase 2 (CK2) phosphoacceptor sites that resembles the SDTD repeats of Mdc1. Furthermore, phosphorylation of this motif is required for interaction with the FHA domain of Nbs1 that localizes Ctp1 to DSB sites. Here, we review and discuss these findings, and we present new data that further characterize the cellular consequences of mutating CK2 phosphorylation motifs of Ctp1, including data showing that these sites are critical for meiosis.

ATR activation necessary but not sufficient for p53 induction and apoptosis in hydroxyurea-hypersensitive myeloid leukemia cells.

Hydroxyurea (HU) is a competitive inhibitor of ribonucleotide reductase that is used for the treatment of myeloproliferative disorders. HU inhibits DNA replication and induces apoptosis in a cell type-dependent manner, yet the relevant pathways that mediate apoptosis in response to this agent are not well characterized. In this study, we employed the human myeloid leukemia 1 (ML-1) cell line as a model to investigate the mechanisms of HU-induced apoptosis. Exposure of ML-1 cells to HU caused rapid cell death that was accompanied by hallmark features of apoptosis, including membrane blebbing, phosphatidylserine translocation, and caspase activation. HU-induced apoptosis required new protein synthesis, was induced by HU exposures as short as 15 min, and correlated with the accumulation of p53 and induction of the p53 target gene PUMA. p53 induction in ML-1 cells was ATR dependent and downregulation of p53 through RNAi delayed HU-induced apoptosis. HU did not induce p53 or induce apoptosis in Molt-3 leukemia cells, even though exposure to HU induced a comparable level of DNA damage and robustly activated the ATR pathway. The microtubule inhibitor nocodazole suppressed HU-induced p53 accumulation in ML-1 cells suggesting that a microtubule-dependent event contributes to p53 induction and apoptosis in this cell line. Our findings outline an HU-induced cell death pathway and suggest that activation of the ATR is necessary, but not sufficient, for stabilization of p53 in response to DNA replication stress.

Absence of Apparent Disease Causing Mutations in COL5A3 in 13 Patients With Hypermobility Ehlers-Danlos Syndrome

Collagen V is a minor fibrillar collagen, broadly distributed as a1(V)2a2(V) heterotrimers [Fessler and Fessler, 1987] that are incorporated into collagen I fibrils, and which regulate the shapes and diameters of collagen I/V heterotypic fibrils [Birk et al., 1988, 1990]. Defects in the COL5A1 and COL5A2 genes underlie at least half of the cases of classic Ehlers–Danlos syndrome (EDS) [Toriello et al., 1996; Wenstrup et al., 1996; Michalickova et al., 1998; Richards et al., 1998; Schwarze et al., 2000; Malfait and De Paepe, 2005]. Collagen V is also found in the form of a relatively uncharacterized a1(V)a2(V)a3(V) heterotrimer, with a limited tissue distribution. Involvement of COL5A1 and COL5A2 defects in classic EDS, andexpressionofa3(V) chains in joint capsule, skin [Brown et al., 1978], and developing ligaments [Imamura et al., 2000] suggested COL5A3 as a candidate locus for the most common form of EDS, the hypermobility type (h-EDS). A candidate locus has yet to be identified for the vast majority of h-EDS cases [Malfait et al., 2006]. Thus, we examined the COL5A3 locus and its products in 13 patients with h-EDS, identified using the previously specified diagnostic criteria [Beighton et al., 1998]. The features of the individual patients are described in Table I. This research was reviewed and approved by the ethics committee of the Hospital for Sick Children. Primer sets (supporting information Table SI may be found in the online version of this article) were designed for amplification and sequencing of COL5A3 genomic DNA and cDNA. RT-PCR primers were designed to allow detection of exon skipping. Dermal fibroblast cultures, established from patients and cultured as described [Chan and Cole, 1991], were grown to confluence prior to RNA and DNA preparation with TRIzol (Invitrogen, Carlsbad, CA). RNA was reverse-transcribed with Superscript II (Invitrogen). Conditions for RT-PCR were 948C for 3 min followed by 35 cycles of 948C for 20 sec, 628C for 40 sec, 728C for 2 min, and final extension at 728C for 10 min. Conditions for amplifying genomic DNA were 948C for 3 min followed by 35 cycles of 948C for 30 sec, 608C for 30 sec, 728C for 3 min, and final extension at 728C for10 min. PCRemployedPromega Taq polymerase. PCR amplimers were purified from 0.8% agarose electrophoresis gels, and direct sequenced using the ABI PRISM BigDye Terminator kit (Applied Biosystems, Foster City, CA). RT-PCR successfully produced COL5A3 cDNA from dermal fibroblasts, although a3(V) expression has not previously been reported for this cell type. Sequencing of cDNA and genomic DNA from the 13 patients with h-EDS showed no apparent diseasecausing mutations typically associated with heritable collagen diseases, such as premature stop codons, exon skipping, or substitutions for obligatory glycines in Gly-X-Y triplets of the major triple helical (COL1) domain [Michalickova et al., 1998; Schwarze et al., 2000; Myllyharju and Kivirikko, 2001]. Analysis of COL5A3 coding sequences identified 11 single nucleotide polymorphisms (SNPs). Two of the 11 SNPs identified in this

Enhanced Tel1ATM Checkpoint Signaling at Protein-Bound Double-Strand Breaks

DNA double-strand breaks (DSBs) pose particularly grave threats to genome integrity and cell survival, as they not only can cause genetic mutations but also can lead to loss of chromosome fragments or genome rearrangements. Just like any structural damage, no two DSBs formed by ionizing radiation or clastogenic agents are exactly alike, and thus the cellular responses that are required to head off a potential catastrophe must be highly adaptable. The ataxia-telangiectasia mutated (ATM) protein kinase controls many of the cellular responses to DSBs in humans (7). In this issue, Fukunaga et al. (4) report that Tel1, theSaccharomyces cerevisiae ortholog of ATM, is hyperactivated when DNA ends are covalently bound to proteins. These unexpected results suggest that the strength of the checkpoint response can be increased by “dirty” DNA ends, which may be more difficult to repair. ATM phosphorylates a plethora of substrates that are involved in control of the cell cycle checkpoint, DNA repair, and apoptosis in higher eukaryotes (7). In humans, defects in the ATM gene can cause neurodegeneration, immunodeficiencies, and severe radiosensitivity. While some of the downstream functions of ATM have been characterized, key aspects of the ATM activation mechanism remain unsolved. ATM exists as a homodimer in its inactive state in vivo (1). Inactive ATM recognizes and interacts with the heterotrimeric Mre11-Rad50Nbs1 (MRN) complex, which binds the DNA ends generated at a DSB. This process, as well as trans autophosphorylation between each partner in the ATM-ATM dimer, generates active ATM monomers. Fukunaga et al. initiated their study by establishing a sensitive assay that measures the activity of immunoprecipitated Tel1. This analysis confirmed that stimulation of Tel1 activity by DSBs relies on its association with the Mre11-Rad50Xrs2 (MRX) protein complex. Furthermore, this interaction requires the C terminus of Xrs2. These findings parallel studies done with human and Xenopus ATM (3, 5, 6), although interestingly, Tel1 recruitment to DSBs does not depend on its kinase activity, representing a significant difference from ATM (2). The MRX protein complex processes DNA ends in association with Sae2 nuclease, which is homologous to human CtIP and fission yeast Ctp1. Together with other enzymes that can catalyze the 5 -to-3 resection of DNA ends, MRX and Sae2 generate the 3 single-strand DNA overhangs that are essential for homologous recombination repair of DSBs. Interestingly, deletion of SAE2 enhances Tel1 function in vivo (10), but the mechanism underlying this effect is unknown. Fukunaga and colleagues show that Sae2 is particularly important for processing DNA ends that are covalently bound to proteins. This was previously shown for removal of Spo11, a topoisomerase-related protein that becomes covalently bound to the 5 end of DSBs formed during meiosis (8). Fukunaga et al. report that Sae2 is also required for the efficient removal of histones that become covalently bound to DNA in cells treated with phleomycin, a chemical clastogen. They detected a similar requirement for Sae2 and Mre11 nuclease activity in the efficient removal of topoisomerase I (Top1), which can be trapped on 3 DNA ends by camptothecin (CPT), a Top1 poison. The defect in Top1 removal in sae2 cells correlated with an enhancement of Tel1 activation. To test whether DNA ends that are covalently bound to proteins might hyperactivate Tel1, in vitro kinase assays were performed with purified Tel1 and MRX complex in the presence of double-stranded DNAs whose ends were prebound with Fab antibody fragments (4). Remarkably, these DNA-Fab fragment complexes stimulated Tel1 activity to a level 2-fold greater than that produced by naked DNA duplexes alone (Fig. 1). Importantly, Fab fragments bound to internal regions of the DNA duplexes had no such effect. As tethering of Fab fragments to DNA ends slows the rate of MRX-mediated nucleolytic DNA end processing, an obvious prediction from the data by Fukunaga et al. was that any method of inhibiting DNA end processing might increase Tel1 activation. They tested this model by repeating the assays using a nuclease-defective form of Mre11 (encoded by mre11-3), which lingers at DSBs due to a defect in DNA end processing. However, this nuclease-defective Mre11 mutant did not enhance Tel1 activation in the presence of protein-free DNA ends. Interestingly, Tel1 kinase activity gradually diminished over time when wild-type MRX complex was incubated with duplex DNA molecules that were blocked at their ends with Fab fragments. This reduction in Tel1 activity was not observed when the experiment was performed with the nuclease-dead Mre11 mutant. These results suggest that Fab fragments do not fully protect DNA ends from MRX-mediated end processing, which eventually converts the DNA molecules into forms that cannot activate Tel1, a process that has also been observed for ATM (9). * Corresponding author. Mailing address: The Scripps Research Institute, Department of Molecular Biology, MB-3, 10550 North Torrey Pines Road, La Jolla, CA 92037. Phone and fax: (858) 784-8273. E-mail: prussell@scripps.edu. Published ahead of print on 28 March 2011.