Defective DNA Ligation during Short-Patch Single-Strand Break Repair in Ataxia Oculomotor Apraxia 1

Ataxia oculomotor apraxia 1 (AOA1) results from mutations in aprataxin, a component of DNA strand break repair that removes AMP from 5′ termini. Despite this, global rates of chromosomal strand break repair are normal in a variety of AOA1 and other aprataxin-defective cells. Here we show that short-patch single-strand break repair (SSBR) in AOA1 cell extracts bypasses the point of aprataxin action at oxidative breaks and stalls at the final step of DNA ligation, resulting in the accumulation of adenylated DNA nicks. Strikingly, this defect results from insufficient levels of nonadenylated DNA ligase, and short-patch SSBR can be restored in AOA1 extracts, independently of aprataxin, by the addition of recombinant DNA ligase. Since adenylated nicks are substrates for long-patch SSBR, we reasoned that this pathway might in part explain the apparent absence of a chromosomal SSBR defect in aprataxin-defective cells. Indeed, whereas chemical inhibition of long-patch repair did not affect SSBR rates in wild-type mouse neural astrocytes, it uncovered a significant defect in Aptx^−/− neural astrocytes. These data demonstrate that aprataxin participates in chromosomal SSBR in vivo and suggest that short-patch SSBR arrests in AOA1 because of insufficient nonadenylated DNA ligase.Oxidative stress is an etiological factor in many neurological diseases, including Alzheimer''s disease, Parkinson''s disease, and Huntington''s disease. One type of macromolecule damaged by reactive oxygen species is DNA, and oxidative damage to DNA has been suggested to be a significant factor in these and other neurological conditions (2). In particular, a number of rare hereditary neurodegenerative disorders have provided direct support for the notion that unrepaired DNA damage causes neural dysfunction. Not least of these are the recessive spinocerebellar ataxias, a number of which are associated with mutations in DNA damage response proteins (17). The archetypal DNA damage-associated spinocerebellar ataxia is ataxia-telangiectasia (A-T), in which mutations in ATM protein result in defects in the detection and signaling of DNA double-strand breaks (DSBs) (3). A-T-like disorder is a related disease that exhibits neurological features similar to those of A-T, resulting from mutation of Mre11, a component of the MRN complex that operates in conjunction with ATM during DSB detection and signaling (28).Two additional spinocerebellar ataxias are spinocerebellar ataxia with axonal neuropathy 1 (SCAN1) and ataxia oculomotor apraxia 1 (AOA1), in which the TDP1 and aprataxin proteins are mutated, respectively (9, 19, 27). Both TDP1 and aprataxin are components of the DNA strand break repair machinery (recently reviewed in references 6 and 24). Whereas SCAN1 is currently limited to nine individuals from a single family, AOA1 is one of the commonest recessive spinocerebellar ataxias. Aprataxin is a member of the histidine triad superfamily of nucleotide hydrolases/transferases and has been reported to remove phosphate and phosphoglycolate moieties from the 3′ termini of DNA strand breaks (26). Aprataxin can also remove AMP from a variety of ligands in vitro, including adenosine polyphosphates, AMP-lysine, AMP-NH₂ (adenine monophosphoramidate), and adenylated DNA in which AMP is covalently attached to the 5′ terminus of a DNA single-strand break (SSB) or DSB (1, 16, 23, 25). To date, aprataxin activity is greatest on AMP-DNA, suggesting that this may be the physiological substrate of this enzyme.In vitro, DNA strand breaks with 5′-AMP termini can arise from premature DNA ligase activity. DNA ligases adenylate 5′ termini at DNA breaks to enable nucleophilic attack of the resulting pyrophosphate bonds by 3′-hydroxyl termini, thereby resealing the breaks. However, DNA adenylation by DNA ligases can occur prematurely, before a 3′-hydroxyl terminus is available. Aprataxin reverses these premature DNA adenylation events, in vitro at least, effectively “resetting” the DNA ligation reaction to the beginning (1). Whether or not 5′-AMP arises in DNA in vivo or is a physiological substrate of aprataxin, however, is unknown. Moreover, attempts to measure DNA strand break repair rates in vivo are conflicting and have failed to identify a consistent defect in DNA SSB repair (SSBR) or DSB repair (DSBR) in AOA1 cells (14, 15, 20). It is thus not clear whether or not defects in DNA strand break repair can account for this neurodegenerative disease.Here we have resolved the discrepancy between the requirements for aprataxin in vitro and in vivo by identifying the stage at which SSBR reactions fail in vitro and by carefully analyzing chromosomal SSBR rates in vivo. We show that short-patch SSBR reactions are defective in AOA1 cell extracts at the final step of DNA ligation, resulting in the accumulation of adenylated DNA nicks, and that this defect can be rescued in AOA1 extracts independently of aprataxin by addition of recombinant DNA ligase. We also find that treatment with aphidicolin, an inhibitor of DNA polymerase δ (Pol δ) and Pol ɛ, unveils a measurable defect in chromosomal SSBR in Aptx^−/− primary neural astrocytes, suggesting that the adenylated nicks that arise from the short-patch repair defect can be channeled into long-patch repair in vivo. These data demonstrate that aprataxin participates in chromosomal SSBR and suggest that this process arrests in AOA1, at oxidative SSBs, due to insufficient levels of nonadenylated DNA ligase.     

The Role of the DNA Damage Response Kinase Ataxia Telangiectasia Mutated in Neuroprotection

It has been estimated that a human cell is confronted with 1 million DNA lesions every day, one fifth of which may originate from the activity of Reactive Oxygen Species (ROS) alone [1,2]. Terminally differentiated neurons are highly active cells with, if any, very restricted regeneration potential [3]. In addition, genome integrity and maintenance during neuronal development is crucial for the organism. Therefore, highly accurate and robust mechanisms for DNA repair are vital for neuronal cells. This requirement is emphasized by the long list of human diseases with neurodegenerative phenotypes, which are either caused by or associated with impaired function of proteins involved in the cellular response to genotoxic stress [4-8]. Ataxia Telangiectasia Mutated (ATM), one of the major kinases of the DNA Damage Response (DDR), is a node that links DDR, neuronal development, and neurodegeneration [2,9-12]. In humans, inactivating mutations of ATM lead to Ataxia-Telangiectasia (A-T) disease [11,13], which is characterized by severe cerebellar neurodegeneration, indicating an important protective function of ATM in the nervous system [14]. Despite the large number of studies on the molecular cause of A-T, the neuroprotective role of ATM is not well established and is contradictory to its general proapoptotic function. This review discusses the putative functions of ATM in neuronal cells and how they might contribute to neuroprotection.     

Quantitative Phosphoproteomics of the Ataxia Telangiectasia-Mutated (ATM) and Ataxia Telangiectasia-Mutated and Rad3-related (ATR) Dependent DNA Damage Response in Arabidopsis thaliana

The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is an important biological regulatory mechanism. In the context of genome integrity, signaling cascades driven by phosphorylation are crucial for the coordination and regulation of DNA repair. The two serine/threonine protein kinases ataxia telangiectasia-mutated (ATM) and Ataxia telangiectasia-mutated and Rad3-related (ATR) are key factors in this process, each specific for different kinds of DNA lesions. They are conserved across eukaryotes, mediating the activation of cell-cycle checkpoints, chromatin modifications, and regulation of DNA repair proteins. We designed a novel mass spectrometry-based phosphoproteomics approach to study DNA damage repair in Arabidopsis thaliana. The protocol combines filter aided sample preparation, immobilized metal affinity chromatography, metal oxide affinity chromatography, and strong cation exchange chromatography for phosphopeptide generation, enrichment, and separation. Isobaric labeling employing iTRAQ (isobaric tags for relative and absolute quantitation) was used for profiling the phosphoproteome of atm atr double mutants and wild type plants under either regular growth conditions or challenged by irradiation. A total of 10,831 proteins were identified and 15,445 unique phosphopeptides were quantified, containing 134 up- and 38 down-regulated ATM/ATR dependent phosphopeptides. We identified known and novel ATM/ATR targets such as LIG4 and MRE11 (needed for resistance against ionizing radiation), PIE1 and SDG26 (implicated in chromatin remodeling), PCNA1, WAPL, and PDS5 (implicated in DNA replication), and ASK1 and HTA10 (involved in meiosis).In eukaryotes, the reversible phosphorylation of serine, threonine, and tyrosine residues within proteins is a wide-spread post-translational modification, essential for controlling a multitude of cellular processes. During the last decade, sequencing projects unexpectedly unraveled that plant genomes encode for a considerable larger number of protein kinases than the other kingdoms of life. Arabidopsis thaliana contains 1112 PKs (4% of all genes), twice the number encoded by the human genome (518 or 2% of all genes) and other plants have an even higher number of kinases (1).Phosphatidyl inositol 3′ kinase related kinases are important players in DNA damage response (DDR)¹ and crucial for genome integrity (2). Key to DNA double strand break (DSB) repair is a chain of events starting with detection of the lesion, activation of a signaling cascade, cell cycle arrest, and recruitment of the repair machinery. The cascade is triggered by the Phosphatidyl inositol 3′ kinase related kinases family kinases ataxia telangiectasia-mutated (ATM) (3) and Ataxia telangiectasia-mutated and Rad3-related (ATR) (4). Both kinases are conserved across eukaryotes. Their downstream targets have been systematically identified in yeast (5) and human cells (6, 7). Their essential role in mediating DNA repair in higher plants has been established (8–10). In Arabidopsis, loss of function mutants are viable (11); however, atm mutants are highly sensitive to genotoxic stress and have a reduced fertility. atr mutant plants have a cell-cycle checkpoint defect upon exposure to genotoxic chemicals (12). Somatic growth under nonchallenging conditions is not affected in the double mutant but plants are sterile, highlighting the role of both kinases coordinating meiotic DNA repair. In plants, systematic phosphoproteomic studies of the involved pathways have not been reported but would contribute to further elucidating the molecular mechanism of the observed phenotypes. Interestingly, plants lack clear homologs for many downstream regulatory components in the signaling cascade (e.g. CHK1, CHK2, p53, and MDC1) (13). In this context, it should be noted that DNA-PKcs (DNA-dependent protein kinase), another Phosphatidyl inositol 3′ kinase related kinases family member involved in DNA repair, has not been identified in plant genomes (14, 15), underscoring the significance of ATM and ATR as master regulators.ATM is recruited to DSBs via its interaction with NBS1/XRS2, a member of the MRN/X complex (MRE11/RAD50/NBS1-XRS2). In plants, the detailed molecular base for ATM recruitment has remained unknown. The complex acts as damage sensor in yeast, first to be detected at DNA double strand break (DSB) sites and essential for resection of DNA (16). In all organisms analyzed, the MRN/X complex is required for genotoxic stress resistance (17). The Mre11 endonuclease activity is critical for ATM activation, likely triggered by the generation of short oligo-nucleotides (18). In higher eukaryotes, ATM activation relies on MRN binding to DSBs via MRE11, subsequent tethering of DSB ends via RAD50 and recruitment of ATM. This interaction leads to monomerisation of inactive ATM dimers, followed by autophosphorylation. The MRN subcomplex member NBS1 interacts with monomeric ATM leading to its localization in close proximity of the DSB site (19). NBS1, H2AX, the checkpoint kinase CHK2, and the trimeric replication protein A (RPA) are important downstream targets of ATM (6).In yeast, ATR is activated by RPA coated single-stranded DNA (ssDNA) that is generated by 5′ resection mediated by MRX/N, Exo1, Sgs1, and Dna2 during DSB processing (20). Furthermore, ssDNA may become exposed because of replication fork break down during DNA replication or nucleotide excision repair (21). Exposed ssDNA is rapidly bound by RPA, attracting ATRIP, and the Rad17-RFC complex. ATRIP interacts with ATR and is essential for its activation and function (22). The Rad17-RFC complex is functional in loading the 9–1-1 protein complex (Rad9, Rad1, and Hus1) to 5′ dsDNA-ssDNA junctions, in turn stimulating ATR activity at the site of the exposed ssDNA (23). In human cells, TopBP1 is required for activation of ATR and localizing to DNA lesion sites. Rad17, TopBP1, RPA, and the checkpoint kinase CHK1 are known downstream targets of activated ATR.The core effectors of DNA repair (e.g. RAD51), the proteins detecting DNA damage and mediating initiation of repair (e.g. MRX) and the two master regulators ATM and ATR are conserved in plants but many downstream components have diverged considerably. Yet, a comprehensive model for DDR in plants requires identification of all components to delineate the involved signaling pathways and their cross-talk with other regulatory processes.Mass spectrometry-based methods are powerful and hypothesis-free approaches for protein characterization, enabling high-throughput studies of protein complexes (24), protein expression profiling (25), or large-scale identification of protein kinase targets (26). Also, the identification and quantification of thousands of phosphopeptides has become feasible by technological and methodological advances. As a consequence, system-wide analyses of signaling networks has become possible (27). Despite above mentioned advances, comprehensive phosphoproteomic studies remain challenging in regards to sample preparation and phosphopeptide enrichment. An additional complication in large-scale studies is imposed by the requirement for correct automatic localization of phosphorylation sites (28). Abundant metabolites make sample preparation in plants especially difficult; however, a number of large-scale phosphoproteomic studies have been reported (29–34).Phosphorylation sites in proteins are in most cases substoichiometric. As a consequence, the comprehensive analysis requires enrichment of phosphopeptides prior to LC-MS/MS. From the large number of developed methods (35), metal-based affinity chromatography such as immobilized metal affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC) have become most widely used. Both materials have different specificities, resulting in a substantial increase of identified phosphopeptides when employed consecutively (36).Here we delineate a methodology to identify and relatively quantify phosphorylation events proteome-wide in higher plants (Fig. 1). We demonstrate its applicability in the context of ATM and ATR dependent DNA damage repair in Arabidopsis thaliana. The approach combines filter assisted sample preparation (FASP) (37), isobaric labeling via iTRAQ, phosphopeptide enrichment using IMAC and TiO₂, strong cation exchange (SCX) chromatography, followed by LC-MS/MS. We compared the relative differences between the phosphoproteomes of wild type plants with the double mutant (atm atr), studying both irradiated and nonirradiated plants. In addition, we performed an independent analysis based on peptide generation after protein precipitation.Open in a separate windowFig. 1.Workflow for identification of ATM/ATR dependent and independent phosphorylations. Wild type and atm atr double mutant plants were either exposed to irradiation or grown under regular conditions. Extracted proteins were purified via FASP and labeled with iTRAQ. Phosphopeptides were enriched by consecutive application of IMAC and TiO₂. Both, the phosphopeptide-enriched fraction and the flow-through of the TiO₂ chromatography, were separated by SCX chromatography and fractions were analyzed by reversed phase LC-MS/MS.All together, we identified 10,831 proteins. Four-hundred and 13 phosphoproteins are phosphorylated upon ionizing radiation, among them 108 in an ATM/ATR dependent manner. The acquired data-set represents a unique resource for plant researchers and extends the current knowledge on ATM/ATR dependent DNA repair pathways.     

Phosphorylation of p53 by TAF1 Inactivates p53-Dependent Transcription in the DNA Damage Response

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Deregulation of Internal Ribosome Entry Site-Mediated p53 Translation in Cancer Cells with Defective p53 Response to DNA Damage

Synthesis of the p53 tumor suppressor and its subsequent activation following DNA damage are critical for its protection against tumorigenesis. We previously discovered an internal ribosome entry site (IRES) at the 5′ untranslated region of the p53 mRNA. However, the connection between IRES-mediated p53 translation and p53''s tumor suppressive function is unknown. In this study, we identified two p53 IRES trans-acting factors, translational control protein 80 (TCP80), and RNA helicase A (RHA), which positively regulate p53 IRES activity. Overexpression of TCP80 and RHA also leads to increased expression and synthesis of p53. Furthermore, we discovered two breast cancer cell lines that retain wild-type p53 but exhibit defective p53 induction and synthesis following DNA damage. The levels of TCP80 and RHA are extremely low in both cell lines, and expression of both proteins is required to significantly increase the p53 IRES activity in these cells. Moreover, we found cancer cells transfected with a shRNA against TCP80 not only exhibit decreased expression of TCP80 and RHA but also display defective p53 induction and diminished ability to induce senescence following DNA damage. Therefore, our findings reveal a novel mechanism of p53 inactivation that links deregulation of IRES-mediated p53 translation with tumorigenesis.     

Causes and Consequences of the DNA Damage Response

A prerequisite for maintaining genome stability in all cell types is the accurate repair and efficient signaling of DNA double strand breaks (DSBs). It is believed that DSBs are initially detected by damage sensors that trigger the activation of transducing kinases. These transducers amplify the damage signal, which is then relayed to effector proteins, which regulate the progression of the cell cycle, DNA repair and apoptosis. Errors in the execution of the repair and/or signaling of DSBs can give rise to multi-systemic disorders characterized by tissue degeneration, infertility, immune system dysfunction, age-related pathologies and cancer. This special Spotlight issue of Cell Cycle highlights recent advances in our understanding of the biology and significance of the DNA damage response. A range of issues are addressed including mechanistic ones: what is the aberrant DNA structure that triggers the activation of the checkpoint - how does chromatin structure influence the recruitment of repair and checkpoint proteins- how does chromosomal instability contribute to the evolution of cancer. In addition, questions related to the physiology of the DNA damage response in normal and abnormal cells is explored: what is the in vivo consequence of altering specific amino acids in a DNA damage sensor- does DNA damage accumulation in stem cells cause aging- how is neurodegeneration linked to deficiencies in specific DNA repair pathways, and finally, what is the biological basis for selection of aberrant DNA damage responses in cancer cells?     

Senataxin, defective in ataxia oculomotor apraxia type 2, is involved in the defense against oxidative DNA damage

A defective response to DNA damage is observed in several human autosomal recessive ataxias with oculomotor apraxia, including ataxia-telangiectasia. We report that senataxin, defective in ataxia oculomotor apraxia (AOA) type 2, is a nuclear protein involved in the DNA damage response. AOA2 cells are sensitive to H2O2, camptothecin, and mitomycin C, but not to ionizing radiation, and sensitivity was rescued with full-length SETX cDNA. AOA2 cells exhibited constitutive oxidative DNA damage and enhanced chromosomal instability in response to H2O2. Rejoining of H2O2-induced DNA double-strand breaks (DSBs) was significantly reduced in AOA2 cells compared to controls, and there was no evidence for a defect in DNA single-strand break repair. This defect in DSB repair was corrected by full-length SETX cDNA. These results provide evidence that an additional member of the autosomal recessive AOA is also characterized by a defective response to DNA damage, which may contribute to the neurodegeneration seen in this syndrome.     

Mitochondria DNA Change and Oxidative Damage in Clinically Stable Patients with Major Depressive Disorder

BackgroundTo compare alterations of mitochondria DNA (mtDNA) copy number, single nucleotide polymorphisms (SNPs), and oxidative damage of mtDNA in clinically stable patients with major depressive disorder (MDD).MethodsPatients met DSM-IV diagnostic criteria for MDD were recruited from the psychiatric outpatient clinic at Changhua Christian Hospital, Taiwan. They were clinically stable and their medications had not changed for at least the preceding two months. Exclusion criteria were substance-induced psychotic disorder, eating disorder, anxiety disorder or illicit substance abuse. Comparison subjects did not have any major psychiatric disorder and they were medically healthy. Peripheral blood leukocytes were analyzed to compare copy number, SNPs and oxidative damage of mtDNA between the two groups.Results40 MDD patients and 70 comparison subjects were collected. The median age of the subjects was 42 years and 38 years in MDD and comparison groups, respectively. Leukocyte mtDNA copy number of MDD patients was significantly lower than that of the comparison group (p = 0.037). MDD patients had significantly higher mitochondrial oxidative damage than the comparison group (6.44 vs. 3.90, p<0.001). After generalized linear model adjusted for age, sex, smoking, family history, and psychotropic use, mtDNA copy number was still significantly lower in the MDD group (p<0.001). MtDNA oxidative damage was positively correlated with age (p<0.001) and MDD (p<0.001). Antipsychotic use was negatively associated with mtDNA copy number (p = 0.036).LimitationsThe study is cross-sectional with no longitudinal follow up. The cohort is clinically stable and generalizability of our result to other cohort should be considered.ConclusionsOur study suggests that oxidative stress and mitochondria may play a role in the pathophysiology of MDD. More large-scale studies are warranted to assess the interplay between oxidative stress, mitochondria dysfunction and MDD.     

TPX2 Impacts Acetylation of Histone H4 at Lysine 16: Implications for DNA Damage Response

During interphase, the spindle assembly factor TPX2 is compartmentalized in the nucleus where its roles remain largely uncharacterized. Recently, we found that TPX2 regulates the levels of serine 139-phosphoryated H2AX (γ-H2AX) at chromosomal breaks induced by ionizing radiation. Here, we report that TPX2 readily associates with the chromatin in the absence of ionizing radiation. Overexpression of TPX2 alters the DAPI staining pattern of interphase cells and depletion of TPX2 constitutively decreases the levels of histone H4 acetylated at lysine16 (H4K16ac) during G1-phase. Upon ionizing irradiation, this constitutive TPX2 depletion-dependent decrease in H4K16ac levels correlates with increased levels of γ-H2AX. The inversely correlated levels of H4K16ac and γ-H2AX can also be modified by altering the levels of SIRT1, herein identified as a novel protein complex partner of TPX2. Furthermore, we find that TPX2 depletion also interferes with formation of 53BP1 ionizing radiation-induced foci, known to depend on γ-H2AX and the acetylation status of H4K16. In brief, our study is the first indication of a constitutive control of TPX2 on H4K16ac levels, with potential implications for DNA damage response.     

DNA损伤反应性蛋白参与中心体调控

中心体是动物细胞有丝分裂期微管组织中心,对于细胞有丝分裂期形成纺锤体、正常分裂及染色体精确分离至关重要. 中心体失调控常造成遗传物质错误分配,最终诱发肿瘤形成.因此,对中心体结构及数量的精密调控将对细胞命运起着决定 作用.目前发现,中心体至少包含100多种调节蛋白,这些蛋白在细胞内的功能各异.最近很多研究显示,多种DNA损伤修复及 应答通路的激酶或磷酸酶定位于中心体,并且参与中心体调控.本文将对中心体结构、中心体复制、中心体分离、中心体扩 增、DNA损伤与中心体异常及DNA损伤反应性蛋白在中心体调控中的功能作一综述.