Nucleoside Diphosphate Kinase Escalates A-to-C Mutations in MutT-Deficient Strains of Escherichia coli.

The chemical integrity of the nucleotide pool and its homeostasis are crucial for genome stability. Nucleoside diphosphate kinase (NDK) is a crucial enzyme that carries out reversible conversions from nucleoside diphosphate (NDP) to nucleoside triphosphate (NTP) and deoxynucleoside diphosphate (dNDP) to deoxynucleoside triphosphate (dNTP). Guanosine nucleotides (GDP, GTP, dGDP, and dGTP) are highly susceptible to oxidative damage to 8-oxo-GDP (8-O-GDP), 8-O-dGTP, 8-O-GTP, and 8-O-dGTP. MutT proteins in cells hydrolyze 8-O-GTP to 8-O-GMP or 8-O-dGTP to 8-O-dGMP to avoid its incorporation in nucleic acids. In Escherichia coli, 8-O-dGTP is also known to be hydrolyzed by RibA (GTP cyclohydrolase II). In this study, we show that E. coli NDK catalyzes the conversion of 8-O-dGDP to 8-O-dGTP or vice versa. However, the rate of NDK-mediated phosphorylation of 8-O-dGDP to 8-O-dGTP is about thrice as efficient as the rate of dephosphorylation of 8-O-dGTP to 8-O-dGDP, suggesting an additive role of NDK in net production of 8-O-dGTP in cells. Consistent with this observation, the depletion of NDK (Δndk) in E. coli ΔmutT or ΔmutT ΔribA strains results in a decrease of A-to-C mutations. These observations suggest that NDK contributes to the physiological load of MutT in E. coliIMPORTANCE Nucleoside diphosphate kinase (NDK), a ubiquitous enzyme, is known for its critical role in homeostasis of cellular nucleotide pools. However, NDK has now emerged as a molecule with pleiotropic effects in DNA repair, protein phosphorylation, gene expression, tumor metastasis, development, and pathogen virulence and persistence inside the host. In this study, we reveal an unexpected role of NDK in genome instability because of its activity in converting 8-O-dGDP to 8-O-dGTP. This observation has important consequences in escalating A-to-C mutations in Escherichia coli The severity of NDK in enhancing these mutations may be higher in the organisms challenged with high oxidative stress, which promotes 8-O-dGDP/8-O-dGTP production.

Valproyl-CoA inhibits the activity of ATP- and GTP-dependent succinate:CoA ligases.

BACKGROUND: Valproic acid (VPA) is an effective antiepileptic drug that may induce progressive microvesicular steatosis. The impairment of mitochondrial function may be an important metabolic effect of VPA treatment with potential adverse consequences. OBJECTIVE: To investigate the influence of VPA on the activity of GTP- and ATP-specific succinate:CoA ligases (G-SUCL and A-SUCL). METHODS: The GTP- and ATP-specific SUCL activities were measured in human fibroblasts in the reverse direction, i.e. the formation of succinyl-CoA. These were assessed at different concentrations of succinate in the presence of VPA, valproyl-CoA and zinc chloride, an established inhibitor of the enzymes. Activities were measured using an optimized HPLC procedure. RESULTS: Valproyl-CoA (1 mM) inhibited the activity of A-SUCL and G-SUCL by 45-55% and 25-50%, respectively. VPA (1 mM) had no influence on the activity of the two enzymes. DISCUSSION: Valproyl-CoA appears to affect the activity of SUCL, especially with the ATP-specific enzyme. Considering the key role of SUCL in the Krebs cycle, interference with its activity might impair the cellular energy status. Moreover, A-SUCL is bound to the nucleoside diphosphate kinase (NDPK), which is responsible for the mitochondrial (deoxy)nucleotide synthesis. An inhibition of A-SUCL might influence the activity of NDPK inducing an imbalance of nucleotides in the mitochondria and eventually mitochondrial DNA depletion. This may account for the potential liver failure associated with valproate therapy, reported in patients with deficiencies within the mitochondrial DNA replicase system such as polymerase gamma 1.

Cellular function and molecular structure of ecto-nucleotidases.

Ecto-nucleotidases play a pivotal role in purinergic signal transmission. They hydrolyze extracellular nucleotides and thus can control their availability at purinergic P2 receptors. They generate extracellular nucleosides for cellular reuptake and salvage via nucleoside transporters of the plasma membrane. The extracellular adenosine formed acts as an agonist of purinergic P1 receptors. They also can produce and hydrolyze extracellular inorganic pyrophosphate that is of major relevance in the control of bone mineralization. This review discusses and compares four major groups of ecto-nucleotidases: the ecto-nucleoside triphosphate diphosphohydrolases, ecto-5'-nucleotidase, ecto-nucleotide pyrophosphatase/phosphodiesterases, and alkaline phosphatases. Only recently and based on crystal structures, detailed information regarding the spatial structures and catalytic mechanisms has become available for members of these four ecto-nucleotidase families. This permits detailed predictions of their catalytic mechanisms and a comparison between the individual enzyme groups. The review focuses on the principal biochemical, cell biological, catalytic, and structural properties of the enzymes and provides brief reference to tissue distribution, and physiological and pathophysiological functions.

NME genes in epithelial morphogenesis.

The NME family of genes encodes highly conserved multifunctional proteins that have been shown to participate in nucleic acid metabolism, energy homeostasis, cell signaling, and cancer progression. Some family members, particularly isoforms 1 and 2, have attracted extensive interests because of their potential anti-metastasis activity. Unfortunately, there have been few consensus mechanistic explanations for this critical function because of the numerous molecular functions ascribed to these proteins, including nucleoside diphosphate kinase, protein kinase, nuclease, transcription factor, growth factor, among others. In addition, different studies showed contradictory prognostic correlations between NME expression levels and tumor progression in clinical samples. Thus, analyses using pliable in vivo systems have become critical for unraveling at least some aspects of the complex functions of this family of genes. Recent works using the Drosophila genetic system have suggested a role for NME in regulating epithelial cell motility and morphogenesis, which has also been demonstrated in mammalian epithelial cell culture. This function is mediated by promoting internalization of growth factor receptors in motile epithelial cells, and the adherens junction components such as E-cadherin and ß-catenin in epithelia that form the tissue linings. Interestingly, NME genes in epithelial cells appear to function in a defined range of expression levels. Either down-regulation or over-expression can perturb epithelial integrity, resulting in different aspects of epithelial abnormality. Such biphasic functions provide a plausible explanation for the documented anti-metastatic activity and the suspected oncogenic function. This review summarizes these recent findings and discusses their implications.

The NM23 family in development.

The NM23 (non-metastatic 23) family is almost universally conserved across all three domains of life: eubacteria, archaea and eucaryotes. Unicellular organisms possess one NM23 ortholog, whilst vertebrates possess several. Gene multiplication through evolution has been accompanied by structural and functional diversification. Many NM23 orthologs are nucleoside diphosphate kinases (NDP kinases), but some more recently evolved members lack NDP kinase activity and/or display other functions, for instance, acting as protein kinases or transcription factors. These members display overlapping but distinct expression patterns during vertebrate development. In this review, we describe the functional differences and similarities among various NM23 family members. Moreover, we establish orthologous relationships through a phylogenetic analysis of NM23 members across vertebrate species, including Xenopus laevis and zebrafish, primitive chordates and several phyla of invertebrates. Finally, we summarize the involvement of NM23 proteins in development, in particular neural development. Carcinogenesis is a process of misregulated development, and NM23 was initially implicated as a metastasis suppressor. A more detailed understanding of the evolution of the family and its role in vertebrate development will facilitate elucidation of the mechanism of NM23 involvement in human cancer.

Measurement of human breast tumor cell-secreted shNDPK-B in a murine breast cancer model suggests its role in metastatic progression.

Human breast cancers metastasize early in tumorigenesis and distant lesions, though dormant are very likely extant at the time of diagnosis and treatment in the majority of cases. Removal of primary tumors by surgeons as an imperative of the current treatment approach, also removes inhibitory factors secreted by the primary tumor that had maintained the dormancy of the metastases. We have identified a factor secreted by human breast cancer cells that supports the formation of blood vessels and may be a principal early factor supporting the growth and development of metastases in human disease. Here we demonstrate for the first time that this factor, secreted (s) human (h) nucleoside diphosphate kinase type B (shNDPK-B), product of the nm23-h2 gene, can be detected specifically with high sensitivity (50 pg/ml; 2.5 pM) in an ELISA assay of our own design. We further demonstrate that shNDPK-B is released into the circulation in immunocompromized mice carrying the human breast carcinoma cell MDA-MB-231. These data support the hypothesis that shNDPK-B may be responsible for the early events in angiogenesis supporting both primary and metastatic tumor growth and development.

YNK1, the yeast homolog of human metastasis suppressor NM23, is required for repair of UV radiation- and etoposide-induced DNA damage.

In humans, NM23-H1 is a metastasis suppressor whose expression is reduced in metastatic melanoma and breast carcinoma cells, and which possesses the ability to inhibit metastatic growth without significant impact on the transformed phenotype. NM23-H1 exhibits three enzymatic activities in vitro, each with potential to maintain genomic stability, a 3'-5' exonuclease and two kinases, nucleoside diphosphate kinase (NDPK), and protein histidine kinase. Herein we have investigated the potential contributions of NM23 proteins to DNA repair in the yeast, Saccharomyces cerevisiae, which contains a single NM23 homolog, YNK1. Ablation of YNK1 delayed repair of UV- and etoposide-induced nuclear DNA damage by 3-6h. However, YNK1 had no impact upon the kinetics of MMS-induced DNA repair. Furthermore, YNK1 was not required for repair of mitochondrial DNA damage. To determine whether the nuclear DNA repair deficit manifested as an increase in mutation frequency, the CAN1 forward assay was employed. An YNK1 deletion was associated with increased mutation rates following treatment with either UV (2.6x) or MMS (1.6 x). Mutation spectral analysis further revealed significantly increased rates of base substitution and frameshift mutations following UV treatment in the ynk1Delta strain. This study indicates a novel role for YNK1 in DNA repair in yeast, and suggests an anti-mutator function that may contribute to the metastasis suppressor function of NM23-H1 in humans.

Molecular cloning and analysis of function of nucleoside diphosphate kinase (NDPK) from the scallop Chlamys farreri.

Nucleoside diphosphate kinase (NDPK) is a key metabolic enzyme that catalyzes the synthesis of non-adenine nucleoside triphosphate (NTP) by transferring the terminal phosphate between nucleoside diphosphate (NDP) and NTP. NDPK regulates a variety of eukaryotic cellular activities including cell proliferation, development, and differentiation. The ndpk cDNA was cloned from the hemocytes of the scallop Chlamys farreri and designated Cf-ndpk. The full-length sequence of Cf-ndpk consists of 715 bp encoding a polypeptide of 153 amino acids with a calculated molecular mass of 16927.52 daltons and pI of 7.64. The mRNA expression and distribution of Cf-ndpk in both bacterially challenged and unchallenged scallops were studied by Northern blotting and in situ hybridization. The results showed that Cf-ndpk transcripts were present in hemocytes, gill, adductor muscle, mantle, digestive gland, foot, and gonad, and the expression level increased in hemocytes after bacterial challenge. Recombinant Cf-NDPK expressed in Escherichia coli could transfer the terminal phosphate between UDP and ATP. The Cf-NDPK protein was present in all tested tissues including foot, adductor muscle, digestive gland, gonad, mantle, gill, and hemolymph. It was up-regulated in hemolymph after bacterial challenge. Taken together, these results suggest that NDPK may play roles in the innate immune response of scallop.

Awd, the homolog of metastasis suppressor gene Nm23, regulates Drosophila epithelial cell invasion.

Border cell migration during Drosophila melanogaster oogenesis is a highly pliable model for studying epithelial to mesenchymal transition and directional cell migration. The process involves delamination of a group of 6 to 10 follicle cells from the epithelium followed by guided migration and invasion through the nurse cell complex toward the oocyte. The guidance cue is mainly provided by the homolog of platelet-derived growth factor/vascular endothelial growth factor family of growth factor, or Pvf, emanating from the oocyte, although Drosophila epidermal growth factor receptor signaling also plays an auxiliary role. Earlier studies implicated a stringent control of the strength of Pvf-mediated signaling since both down-regulation of Pvf and overexpression of active Pvf receptor (Pvr) resulted in stalled border cell migration. Here we show that the metastasis suppressor gene homolog Nm23/awd is a negative regulator of border cell migration. Its down-regulation allows for optimal spatial signaling from two crucial pathways, Pvr and JAK/STAT. Its overexpression in the border cells results in stalled migration and can revert the phenotype of overexpressing constitutive Pvr or dominant-negative dynamin. This is a rare example demonstrating the relevance of a metastasis suppressor gene function utilized in a developmental process involving cell invasion.

Crystallographic and biochemical analysis of rotavirus NSP2 with nucleotides reveals a nucleoside diphosphate kinase-like activity.

Rotavirus, the major pathogen of infantile gastroenteritis, carries a nonstructural protein, NSP2, essential for viroplasm formation and genome replication/packaging. In addition to RNA-binding and helix-destabilizing properties, NSP2 exhibits nucleoside triphosphatase activity. A conserved histidine (H225) functions as the catalytic residue for this enzymatic activity, and mutation of this residue abrogates genomic double-stranded RNA synthesis without affecting viroplasm formation. To understand the structural basis of the phosphatase activity of NSP2, we performed crystallographic analyses of native NSP2 and a functionally defective H225A mutant in the presence of nucleotides. These studies showed that nucleotides bind inside a cleft between the two domains of NSP2 in a region that exhibits structural similarity to ubiquitous cellular HIT (histidine triad) proteins. Only minor conformational alterations were observed in the cleft upon nucleotide binding and hydrolysis. This hydrolysis involved the formation of a stable phosphohistidine intermediate. These observations, reminiscent of cellular nucleoside diphosphate (NDP) kinases, prompted us to investigate whether NSP2 exhibits phosphoryl-transfer activity. Bioluminometric assay showed that NSP2 exhibits an NDP kinase-like activity that transfers the bound phosphate to NDPs. However, NSP2 is distinct from the highly conserved cellular NDP kinases in both its structure and catalytic mechanism, thus making NSP2 a potential target for antiviral drug design. With structural similarities to HIT proteins, which are not known to exhibit NDP kinase activity, NSP2 represents a unique example among structure-activity relationships. The newly observed phosphoryl-transfer activity of NSP2 may be utilized for homeostasis of nucleotide pools in viroplasms during genome replication.

Nm23-H1 suppresses tumor cell motility by down-regulating the lysophosphatidic acid receptor EDG2.

Exogenous overexpression of the metastasis suppressor gene Nm23-H1 reduces the metastatic potential of multiple types of cancer cells and suppresses in vitro tumor cell motility and invasion. Mutational analysis of Nm23-H1 revealed that substitution mutants P96S and S120G did not inhibit motility and invasion. To elucidate the molecular mechanism of Nm23-H1 motility suppression, expression microarray analysis of an MDA-MB-435 cancer cell line overexpressing wild-type Nm23-H1 was done and cross-compared with expression profiles from lines expressing the P96S and S120G mutants. Nine genes, MET, PTN, SMO, FZD1, L1CAM, MMP2, NETO2, CTGF, and EDG2, were down-regulated by wild-type but not by mutant Nm23-H1 expression. Reduced expression of these genes coincident with elevated Nm23-H1 expression was observed in human breast tumor cohorts, a panel of breast carcinoma cell lines, and hepatocellular carcinomas from control versus Nm23-M1 knockout mice. The functional significance of the down-regulated genes was assessed by transfection and in vitro motility assays. Only EDG2 overexpression significantly restored motility to Nm23-H1-suppressed cancer cells, enhancing motility by 60-fold in these cells. In addition, silencing EDG2 expression with small interfering RNA reduced the motile phenotype of metastatic breast cancer cells. These data suggest that Nm23-H1 suppresses metastasis, at least in part, through down-regulation of EDG2 expression.

The role of Ureaplasma nucleoside monophosphate kinases in the synthesis of nucleoside triphosphates.

Mollicutes are wall-less bacteria and cause various diseases in humans, animals and plants. They have the smallest genomes with low G + C content and lack many genes of DNA, RNA and protein precursor biosynthesis. Nucleoside diphosphate kinase (NDK), a house-keeping enzyme that plays a critical role in the synthesis of nucleic acids precursors, i.e. NTPs and dNTPs, is absent in all the Mollicutes genomes sequenced to date. Therefore, it would be of interest to know how Mollicutes synthesize dNTPs/NTPs without NDK. To answer this question, nucleoside monophosphate kinases (NMPKs) from Ureaplasma were studied regarding their role in the synthesis of NTPs/dNTPs. In this work, Ureaplasma adenylate kinase, cytidylate kinase, uridylate kinase and thymidylate kinase were cloned and expressed in Escherichia coli. The recombinant enzymes were purified and characterized. These NMPKs are base specific, as indicated by their names, and capable of converting (d)NMPs directly to (d)NTPs. The catalytic rates of (d)NTPs and (d)NDP synthesis by these NMPKs were determined using tritium-labelled (d)NMPs, and the rates for (d)NDP synthesis, in general, were much higher (up to 100-fold) than that of (d)NTP. Equilibrium studies with adenylate kinase suggested that the rates of NTPs/dNTPs synthesis by NMPKs in vivo are probably regulated by the levels of (d)NMPs. These results strongly indicate that NMPKs could substitute the NDK function in vivo.

Double mutant P96S/S120G of Nm23-H1 abrogates its NDPK activity and motility-suppressive ability.

The Nm23-H1 gene is a metastasis suppressor gene. However, its biochemical mechanism of suppressing the metastatic potential of cancer cells is still unknown. The previous hypothesis that a histidine protein kinase activity may contributes to the motility-suppressive effect of Nm23-H1 could not explain why the H118F mutant, a kinase-deficient mutant, still had motility-suppressive ability. We conducted a study on the double mutant P96S/S120G of Nm23-H1 and succeeded in introducing the RP-HPLC method in NDPK assay. The results showed that the double mutant P96S/S120G, when expressed in the bacteria, was completely aggregated in inclusion bodies; this mutant abrogated not only its motility-suppressive ability, but also its NDPK activity. Based on previous work and this study, we prompted that the deficiency of motility-suppressive function of S120G, P96S, and P96S/S120G mutants was due to their altered structure, which might deprive Nm23-H1 of most activities including kinase activity or interactions with other proteins.

Regulation of cardiac cAMP synthesis and contractility by nucleoside diphosphate kinase B/G protein beta gamma dimer complexes.

Heterotrimeric G proteins are pivotal regulators of myocardial contractility. In addition to the receptor-induced GDP/GTP exchange, G protein alpha subunits can be activated by a phosphate transfer via a plasma membrane-associated complex of nucleoside diphosphate kinase B (NDPK B) and G protein betagamma-dimers (Gbetagamma). To investigate the physiological role of this phosphate transfer in cardiomyocytes, we generated a Gbeta1gamma2-dimer carrying a single amino acid exchange at the intermediately phosphorylated His-266 in the beta1 subunit (Gbeta1H266Lgamma2). Recombinantly expressed Gbeta1H266Lgamma2 were integrated into heterotrimeric G proteins in rat cardiomyocytes but were deficient in intermediate Gbeta phosphorylation. Compared with wild-type Gbeta1gamma2 (Gbeta1WTgamma2), overexpression of Gbeta1H266Lgamma2 suppressed basal cAMP formation up to 55%. A similar decrease in basal cAMP production occurred when the formation of NDPK B/Gbetagamma complexes was attenuated by siRNA-mediated NDPK B knockdown. In adult rat cardiomyocytes expressing Gbeta1H266Lgamma2, the basal contractility was suppressed by approximately 50% which correlated to similarly reduced basal cAMP levels and reduced Ser16-phosphorylation of phospholamban. In the presence of the beta-adrenoceptor agonist isoproterenol, the total cAMP formation and contractility were significantly lower in Gbeta1H266Lgamma2 than in Gbeta1WTgamma2 expressing cardiomyocytes. However, the relative isoproterenol-induced increased was not affected by Gbeta1H266Lgamma2. We conclude that the receptor-independent activation of G proteins via NDPK B/Gbetagamma complexes requires the intermediate phosphorylation of G protein beta subunits at His-266. Our results highlight the histidine kinase activity of NDPK B for Gbeta and demonstrate its contribution to the receptor-independent regulation of cAMP synthesis and contractility in intact cardiomyocytes.

NM23-H1 tumor suppressor physically interacts with serine-threonine kinase receptor-associated protein, a transforming growth factor-beta (TGF-beta) receptor-interacting protein, and negatively regulates TGF-beta signaling.

NM23-H1 is a member of the NM23/NDP kinase gene family and a putative metastasis suppressor. Previously, a screen for NM23-H1-interacting proteins that could potentially modulate its activity identified serine-threonine kinase receptor-associated protein (STRAP), a transforming growth factor (TGF)-beta receptor-interacting protein. Through the use of cysteine to serine amino acid substitution mutants of NM23-H1 (C4S, C109S, and C145S) and STRAP (C152S, C270S, and C152S/C270S), we demonstrated that the association between these two proteins is dependent on Cys(145) of NM23-H1 and Cys(152) and Cys(270) of STRAP but did not appear to involve Cys(4) and Cys(109) of NM23-H1, suggesting that a disulfide linkage involving Cys(145) of NM23-H1 and Cys(152) or Cys(270) of STRAP mediates complex formation. The interaction was dependent on the presence of dithiothreitol or beta-mercaptoethanol but not H(2)O(2). Ectopic expression of wild-type NM23-H1, but not NM23-H1(C145S), negatively regulated TGF-beta signaling in a dose-dependent manner, enhanced stable association between the TGF-beta receptor and Smad7, and prevented nuclear translocation of Smad3. Similarly, wild-type NM23-H1 inhibited TGF-beta-induced apoptosis and growth inhibition, whereas NM23-H1(C145S) had no effect. Knockdown of NM23-H1 by small interfering RNA stimulated TGF-beta signaling. Coexpression of wild-type STRAP, but not STRAP(C152S/C270S), significantly stimulated NM23-H1-induced growth of HaCaT cells. These results suggest that the direct interaction of NM23-H1 and STRAP is important for the regulation of TGF-beta-dependent biological activity as well as NM23-H1 activity.

P2y receptor-mediated angiogenesis via vascular endothelial growth factor receptor 2 signaling.

Pathological as well as physiological angiogenesis is known to be regulated by such factors as nucleotides and Vascular Endothelial Growth Factor (VEGF). Activated P2Y nucleotide receptors have been observed to associate and transactivate VEGF Receptor 2 (VEGFR2), suggesting a cooperation between nucleotide and VEGF signaling in angiogenesis. P2YR mediated VEGFR2 signaling therefore may be important in describing the angiogenic signaling of nucleotides such as ATP. Here, we provide evidence that supports the notion of P2YR-VEGFR2 signaling. The significant angiogenic effect of P2Y1/2 receptor agonists (100 microM ATP and 10 microM 2MS-ATP) on endothelial cell tubulogenesis was suppressed back to near control levels upon addition of 1 microM SU1498 (specific VEGFR2 tyrosine kinase inhibitor). We believe that this P2YR-VEFGR2 signaling is an important component of pathological, as well as physiological angiogenesis.

Novel roles of NM23 proteins in skin homeostasis, repair and disease.

Keratinocyte growth factor (KGF) is an important regulator of epidermal homeostasis and repair. Therefore, the identification of KGF target genes in keratinocytes should contribute to our understanding of the molecular mechanisms underlying these processes. In a search for KGF-regulated genes, we identified the gene encoding the nucleoside diphosphate kinase NM23-H1. Apart from a housekeeping function, NM23 proteins are involved in the regulation of many cellular processes as well as in tumor metastasis, but their functions in epidermal homeostasis and repair are largely unknown. Here, we show a high expression of NM23-H1 and NM23-H2 in the KGF-responsive keratinocytes of the hyperproliferative epidermis of mouse skin wounds and of patients suffering from the skin disease psoriasis. To determine if this overexpression is functionally important, we generated HaCaT keratinocyte cell lines overexpressing NM23-H1 and/or -H2. Whereas the enhanced levels of NM23 did not affect cell proliferation in monoculture, NM23-H2 and double transfectants but not NM23-H1 transfectants formed a strongly hyperthickened epithelium in three-dimensional organotypic cultures. The abnormal epithelial morphology resulted from enhanced proliferation, reduced apoptosis and alterations in the differentiation pattern. These findings suggest that epidermal homeostasis depends on a tight regulation of the levels of NM23 isoforms.

The exonuclease TREX1 is in the SET complex and acts in concert with NM23-H1 to degrade DNA during granzyme A-mediated cell death.

Granzyme A (GzmA) activates a caspase-independent cell death pathway with morphological features of apoptosis. Single-stranded DNA damage is initiated when the endonuclease NM23-H1 becomes activated to nick DNA after granzyme A cleaves its inhibitor, SET. SET and NM23-H1 reside in an endoplasmic reticulum-associated complex (the SET complex) that translocates to the nucleus in response to superoxide generation by granzyme A. We now find the 3'-to-5' exonuclease TREX1, but not its close homolog TREX2, in the SET complex. TREX1 binds to SET and colocalizes and translocates with the SET complex. NM23-H1 and TREX1 work in concert to degrade DNA. Silencing NM23-H1 or TREX1 inhibits DNA damage and death of cells treated with perforin (PFN) and granzyme A, but not of cells treated with perforin and granzyme B (GzmB). After granzyme A activates NM23-H1 to make single-stranded nicks, TREX1 removes nucleotides from the nicked 3' end to reduce the possibility of repair by rejoining the nicked ends.