R-loops and impaired autophagy trigger cGAS-dependent inflammation via micronuclei formation in Senataxin-deficient cells.

Senataxin is an evolutionarily conserved DNA/RNA helicase, whose dysfunctions are linked to neurodegeneration and cancer. A main activity of this protein is the removal of R-loops, which are nucleic acid structures capable to promote DNA damage and replication stress. Here we found that Senataxin deficiency causes the release of damaged DNA into extranuclear bodies, called micronuclei, triggering the massive recruitment of cGAS, the apical sensor of the innate immunity pathway, and the downstream stimulation of interferon genes. Such cGAS-positive micronuclei are characterized by defective membrane envelope and are particularly abundant in cycling cells lacking Senataxin, but not after exposure to a DNA breaking agent or in absence of the tumor suppressor BRCA1 protein, a partner of Senataxin in R-loop removal. Micronuclei with a discontinuous membrane are normally cleared by autophagy, a process that we show is impaired in Senataxin-deficient cells. The formation of Senataxin-dependent inflamed micronuclei is promoted by the persistence of nuclear R-loops stimulated by the DSIF transcription elongation complex and the engagement of EXO1 nuclease activity on nuclear DNA. Coherently, high levels of EXO1 result in poor prognosis in a subset of tumors lacking Senataxin expression. Hence, R-loop homeostasis impairment, together with autophagy failure and unscheduled EXO1 activity, elicits innate immune response through micronuclei formation in cells lacking Senataxin.

Sex specific regulation of TSPY-Like 2 in the DNA damage response of cancer cells.

Females have a lower probability to develop somatic cancers and a better response to chemotherapy than males. However, the reasons for these differences are still not well understood. The X-linked gene TSPY-Like 2 (TSPYL2) encodes for a putative tumor suppressor protein involved in cell cycle regulation and DNA damage response (DDR) pathways. Here, we demonstrate that in unstressed conditions TSPYL2 is maintained at low levels by MDM2-dependent ubiquitination and proteasome degradation. Upon genotoxic stress, E2F1 promotes TSPYL2 expression and protein accumulation in non-transformed cell lines. Conversely, in cancer cells, TSPYL2 accumulates only in females or in those male cancer cells that lost the Y-chromosome during the oncogenic process. Hence, we demonstrate that while TSPYL2 mRNA is induced in all the tested tumor cell lines after DNA damage, TSPYL2 protein stability is increased only in female cancer cells. Indeed, we found that TSPYL2 accumulation, in male cancer cells, is prevented by the Y-encoded protein SRY, which modulates MDM2 protein levels. In addition, we demonstrated that TSPYL2 accumulation is required to sustain cell growth arrest after DNA damage, possibly contributing to protect normal and female cancer cells from tumor progression. Accordingly, TSPYL2 has been found more frequently mutated in female-specific cancers. These findings demonstrate for the first time a sex-specific regulation of TSPYL2 in the DDR of cancer cells and confirm the existence of sexual dimorphism in DNA surveillance pathways.

Sex disparities in DNA damage response pathways: Novel determinants in cancer formation and therapy.

Cancer incidence and survival are different between men and women. Indeed, females have a lesser risk and a better prognosis than males in many tumors unrelated to reproductive functions. Although the reasons for these disparities are still unknown, they constitute an important starting point for the development of personalized cancer therapies. One of the mechanisms that fuels carcinogenesis is the accumulation of defects in DNA damage response (DDR) pathways, a complex signaling cascade that senses DNA lesions and, depending on the severity, coordinates transient cell-cycle arrest, DNA replication, repair, apoptosis, and senescence, preventing genomic instability and cancer. Recently, evidence of sexual dimorphisms is emerging in these pathways, therefore providing new opportunities for precision medicine. Here, we will discuss current knowledge about sexual disparities in the DDR, their role in tumorigenesis and cancer progression, and the importance of considering sex contribution in both research and cancer therapies.

Targeting SMYD3 to Sensitize Homologous Recombination-Proficient Tumors to PARP-Mediated Synthetic Lethality.

SMYD3 is frequently overexpressed in a wide variety of cancers. Indeed, its inactivation reduces tumor growth in preclinical in vivo animal models. However, extensive characterization in vitro failed to clarify SMYD3 function in cancer cells, although confirming its importance in carcinogenesis. Taking advantage of a SMYD3 mutant variant identified in a high-risk breast cancer family, here we show that SMYD3 phosphorylation by ATM enables the formation of a multiprotein complex including ATM, SMYD3, CHK2, and BRCA2, which is required for the final loading of RAD51 at DNA double-strand break sites and completion of homologous recombination (HR). Remarkably, SMYD3 pharmacological inhibition sensitizes HR-proficient cancer cells to PARP inhibitors, thereby extending the potential of the synthetic lethality approach in human tumors.

TSPYL2 is a novel regulator of SIRT1 and p300 activity in response to DNA damage.

Protein acetylation and deacetylation events are finely regulated by lysine-acetyl-transferases and lysine-deacetylases and constitute an important tool for the activation or inhibition of specific cellular pathways. One of the most important lysine-acetyl-transferases is p300, which is involved in the regulation of gene expression, cell growth, DNA repair, differentiation, apoptosis, and tumorigenesis. A well-known target of p300 is constituted by the tumor suppressor protein p53, which plays a critical role in the maintenance of genomic stability and whose activity is known to be controlled by post-translational modifications, among which acetylation. p300 activity toward p53 is negatively regulated by the NAD-dependent deacetylase SIRT1, which deacetylates p53 preventing its transcriptional activation and the induction of p53-dependent apoptosis. However, the mechanisms responsible for p53 regulation by p300 and SIRT1 are still poorly understood. Here we identify the nucleosome assembly protein TSPY-Like 2 (TSPYL2, also known as TSPX, DENTT, and CDA1) as a novel regulator of SIRT1 and p300 function. We demonstrate that, upon DNA damage, TSPYL2 inhibits SIRT1, disrupting its association with target proteins, and promotes p300 acetylation and activation, finally stimulating p53 acetylation and p53-dependent cell death. Indeed, in response to DNA damage, cells silenced for TSPYL2 were found to be defective in p53 activation and apoptosis induction and these events were shown to be dependent on SIRT1 and p300 function. Collectively, our results shed new light on the regulation of p53 acetylation and activation and reveal a novel TSPYL2 function with important implications in cancerogenesis.

Cell cycle and apoptosis regulator 2 at the interface between DNA damage response and cell physiology.

Cell cycle and apoptosis regulator 2 (CCAR2 or DBC1) is a human protein recently emerged as a novel and important player of the DNA damage response (DDR). Indeed, upon genotoxic stress, CCAR2, phosphorylated by the apical DDR kinases ATM and ATR, increases its binding to the NAD+-dependent histone deacetylase SIRT1 and inhibits SIRT1 activity. This event promotes the acetylation and activation of p53, a SIRT1 target, and the subsequent induction of p53 dependent apoptosis. In addition, CCAR2 influences DNA repair pathway choice and promotes the chromatin relaxation necessary for the repair of heterochromatic DNA lesions. However, besides DDR, CCAR2 is involved in several other cellular functions. Indeed, through the interaction with transcription factors, nuclear receptors, epigenetic modifiers and RNA polymerase II, CCAR2 regulates transcription and transcript elongation. Moreover, promoting Rev-erbα protein stability and repressing BMAL1 and CLOCK expression, it was reported to modulate the circadian rhythm. Through SIRT1 inhibition, CCAR2 is also involved in metabolism control and, suppressing RelB and p65 activities in the NFkB pathway, it restricts B cell proliferation and immunoglobulin production. Notably, CCAR2 expression is deregulated in several tumors and, compared to the non-neoplastic counterpart, it may be up- or down-regulated. Since its up-regulation in cancer patients is usually associated with poor prognosis and its depletion reduces cancer cell growth in vitro, CCAR2 was suggested to act as a tumor promoter. However, there is also evidence that CCAR2 functions as a tumor suppressor and therefore its role in cancer formation and progression is still unclear. In this review we discuss CCAR2 functions in the DDR and its multiple biological activities in unstressed cells.

A novel crosstalk between CCAR2 and AKT pathway in the regulation of cancer cell proliferation.

Human CCAR2 has recently emerged as having a pivotal role in the DNA damage response, promoting apoptosis and repair of heterochromatic DNA breaks. However, less is known about the function of CCAR2 in tumor formation and cancer progression. Here, we demonstrate, for the first time, that CCAR2 loss inhibits the proliferation of cancer cells, but preserves the growth of normal cells. Investigating the mechanisms responsible for this differential effect, we found that CCAR2 depletion specifically impairs the activation of AKT pathway in cancer cells, but not in normal cells, by reducing AKT phosphorylation on Ser473. This effect is achieved through the transcriptional upregulation of TRB3 gene and accumulation of TRB3 protein, which then binds to and inhibits the phosphorylation and activation of AKT. The defective activation of AKT finally results in reduced GSK3ß phosphorylation, prevention of G1/S transition and inhibition of cancer cell growth. These results establish an important role for CCAR2 in cancer cells proliferation and could shed new light on novel therapeutic strategies against cancer, devoid of detrimental side effects.

Retina-derived POU domain factor 1 coordinates expression of genes relevant to renal and neuronal development.

Retina-derived POU domain Factor 1 (RPF-1), a member of POU transcription factor family, is encoded by POU6F2 gene, addressed by interstitial deletions at chromosome 7p14 in Wilms tumor (WT). Its expression has been detected in developing kidney and nervous system, suggesting an early role for this gene in regulating development of these organs. To investigate into its functions and determine its role in transcriptional regulation, we generated an inducible stable transfectant from HEK293 cells. RPF-1 showed nuclear localization, elevated stability, and transactivation of promoters featuring POU consensus sites, and led to reduced cell proliferation and in vivo tumor growth. By addressing the whole transcriptome regulated by its induction, we could detect a gross alteration of gene expression that is consistent with promoter occupancy predicted by genome-wide Chip-chip analysis. Comparison of bound regulatory regions with differentially expressed genes allowed identification of 217 candidate targets. Enrichment of divergent octamers in predicted regulatory regions revealed promiscuous binding to bipartite POUS and POUH consensus half-sites with intervening spacers. Gel-shift competition assay confirmed the specificity of RPF-1 binding to consensus motifs, and demonstrated that the Ser-rich region upstream of the POU domain is indispensable to achieve DNA-binding. Promoter-reporter activity addressing a few target genes indicated a dependence by RPF-1 on transcriptional response. In agreement with its expression in developing kidney and nervous system, the induced transcriptome appears to indicate a function for this protein in early renal differentiation and neuronal cell fate, providing a resource for understanding its role in the processes thereby regulated.

CCAR2/DBC1 is required for Chk2-dependent KAP1 phosphorylation and repair of DNA damage.

Cell cycle and apoptosis regulator 2 (CCAR2, formerly known as DBC1) is a nuclear protein largely involved in DNA damage response, apoptosis, metabolism, chromatin structure and transcription regulation. Upon DNA lesions, CCAR2 is phosphorylated by the apical kinases ATM/ATR and this phosphorylation enhances CCAR2 binding to SIRT1, leading to SIRT1 inhibition, p53 acetylation and p53-dependent apoptosis. Recently, we found that also the checkpoint kinase Chk2 and the proteasome activator REGγ are required for efficient CCAR2-mediated inhibition of SIRT1 and induction of p53-dependent apoptosis.Here, we report that CCAR2 is required for the repair of heterochromatic DNA lesions, as cells knock-out for CCAR2 retain, at late time-points after genotoxic treatment, abnormal levels of DNA damage-associated nuclear foci, whose timely resolution is reinstated by HP1ß depletion. Conversely, repair of DNA damages in euchromatin are not affected by CCAR2 absence.We also report that the impairment in heterochromatic DNA repair is caused by defective Chk2 activation, detectable in CCAR2 ablated cells, which finally impacts on the phosphorylation of the Chk2 substrate KAP1 that is required for the induction of heterochromatin relaxation and DNA repair.These studies further extend and confirm the role of CCAR2 in the DNA damage response and DNA repair and illustrate a new mechanism of Chk2 activity regulation. Moreover, the involvement of CCAR2 in the repair of heterochromatic DNA breaks suggests a new role for this protein in the maintenance of chromosomal stability, which is necessary to prevent cancer formation.

CHK2 kinase in the DNA damage response and beyond.

The serine/threonine kinase CHK2 is a key component of the DNA damage response. In human cells, following genotoxic stress, CHK2 is activated and phosphorylates >20 proteins to induce the appropriate cellular response, which, depending on the extent of damage, the cell type, and other factors, could be cell cycle checkpoint activation, induction of apoptosis or senescence, DNA repair, or tolerance of the damage. Recently, CHK2 has also been found to have cellular functions independent of the presence of nuclear DNA lesions. In particular, CHK2 participates in several molecular processes involved in DNA structure modification and cell cycle progression. In this review, we discuss the activity of CHK2 in response to DNA damage and in the maintenance of the biological functions in unstressed cells. These activities are also considered in relation to a possible role of CHK2 in tumorigenesis and, as a consequence, as a target of cancer therapy.

Chk2 and REGγ-dependent DBC1 regulation in DNA damage induced apoptosis.

Human DBC1 (Deleted in Breast Cancer 1; KIAA1967; CCAR2) is a protein implicated in the regulation of apoptosis, transcription and histone modifications. Upon DNA damage, DBC1 is phosphorylated by ATM/ATR on Thr454 and this modification increases its inhibitory interaction with SIRT1, leading to p53 acetylation and p53-dependent apoptosis. Here, we report that the inhibition of SIRT1 by DBC1 in the DNA damage response (DDR) also depends on Chk2, the transducer kinase that is activated by ATM upon DNA lesions and contributes to the spreading of DNA damage signal. Indeed we found that inactivation of Chk2 reduces DBC1-SIRT1 binding, thus preventing p53 acetylation and DBC1-induced apoptosis. These events are mediated by Chk2 phosphorylation of the 11S proteasome activator REGγ on Ser247, which increases REGγ-DBC1 interaction and SIRT1 inhibition. Overall our results clarify the mechanisms underlying the DBC1-dependent SIRT1 inhibition and link, for the first time, Chk2 and REGγ to the ATM-DBC1-SIRT1 axis.

DBC1 phosphorylation by ATM/ATR inhibits SIRT1 deacetylase in response to DNA damage.

Human DBC1 (deleted in breast cancer-1; KIAA1967) is a nuclear protein that, in response to DNA damage, competitively inhibits the NAD(+)-dependent deacetylase SIRT1, a regulator of p53 apoptotic functions in response to genotoxic stress. DBC1 depletion in human cells increases SIRT1 activity, resulting in the deacetylation of p53 and protection from apoptosis. However, the mechanisms regulating this process have not yet been determined. Here, we report that, in human cell lines, DNA damage triggered the phosphorylation of DBC1 on Thr454 by ATM (ataxia telangiectasia-mutated) and ATR (ataxia telangiectasia and Rad3-related) kinases. Phosphorylated DBC1 bound to and inhibited SIRT1, resulting in the dissociation of the SIRT1-p53 complex and stimulating p53 acetylation and p53-dependent cell death. Indeed, DBC1-mediated genotoxicity, which was shown in knockdown experiments to be dependent on SIRT1 and p53 expression, was defective in cells expressing the phospho-mutant DBC1(T454A). This study describes the first post-translational modification of DBC1 and provides new mechanistic insight linking ATM/ATR to the DBC1-SIRT1-p53 apoptotic axis triggered by DNA damage.

A protein phosphatase feedback mechanism regulates the basal phosphorylation of Chk2 kinase in the absence of DNA damage.

The checkpoint kinase Chk2 is an effector component of the ATM-dependent DNA damage response (DDR) pathway. The activation of Chk2 by genotoxic stress involves its phosphorylation on T68 by ATM and additional auto/transphosphorylations. Here we demonstrate that in unperturbed cells, chemical inhibition of Chk2 by VRX0466617 (VRX) enhances the phosphorylation of Chk2-T68 throughout the cell cycle phases. This event, dependent on the presence of ATM and catalytically functional Chk2, is not consequential to DNA damage, as neither gamma-H2AX nuclear foci nor increased ATM activation is detected in VRX-treated cells, suggesting the involvement of other regulatory proteins. As serine/threonine protein phosphatases (PPs) regulate the phosphorylation and deactivation of proteins of the DDR pathway, we analyzed their role in phospho-T68-Chk2 regulation. We found that intracellular inhibition of PP1 and PP2A-like activities by okadaic acid markedly raised the accumulation of Chk2-pT68 without DNA damage induction, and this phenomenon was also seen when PP1-C, PP2A-C, and Wip1/PPM1D were simultaneously knockdown by siRNA. Altogether, these data indicate a novel mechanism in undamaged cells where PPs function to maintain the balance between ATM and its direct substrate Chk2 through a regulatory circuit.

REGgamma/PA28gamma proteasome activator interacts with PML and Chk2 and affects PML nuclear bodies number.

REGgamma (also called PA28gamma or PSME3) is a proteasome activator involved in the degradation of several proteins that regulate cell cycle and transcription. Recently, we demonstrated that this protein has a role also in the maintenance of chromosomal stability and in the response to spindle damaging agents. Here we report for the first time that REGgamma interacts with the promyelocytic leukemia protein (PML), accumulates in PML nuclear bodies (PML-NBs), but it does not play any role in normal or arsenic-induced PML degradation. However, REGgamma seems to regulate PML-NBs number, since its deficiency causes an increase in PML-NBs, which can be overcome by increased levels of SUMO1, and its overexpression has the opposite effect. We additionally found that REGgamma interacts with the DNA damage checkpoint kinase Chk2, whose presence is necessary for the increase of PML-NBs induced by REGgamma deficiency, and that REGgamma depletion resulted in a partial restoration of PML-NBs in APL derived cells. Altogether, these results underline a new role for REGgamma in the control and regulation of PML subnuclear structures.

The shelterin protein TRF2 inhibits Chk2 activity at telomeres in the absence of DNA damage.

The shelterin complex [1] shapes and protects telomeric DNA from being processed as double strand breaks (DSBs) [2, 3]. Here we show that in human undamaged cells, a fraction of the kinase Chk2, a downstream target of ATM and mediator of checkpoint responses and senescence [4, 5], physically interacts with the shelterin subunit TRF2 and colocalizes with this complex at chromosome ends. This interaction, enhanced by TRF2 binding to telomeric DNA, inhibits the activation and senescence-induced function of Chk2 by a mechanism in which TRF2 binding to the N terminus of Chk2 surrounding Thr68 hinders the phosphorylation of this priming site. In response to radiation-induced DSBs, but not chromatin-remodelling agents, the telomeric Chk2-TRF2 binding dissociates in a Chk2 activity-dependent manner. Moreover, active Chk2 phosphorylates TRF2 and decreases its binding to telomeric DNA repeats, corroborating the evidences on the specific TRF2 relocalization in presence of DSBs [6]. Altogether, the capacity of TRF2 to locally repress Chk2 provides an additional level of control by which shelterin restrains the DNA damage response from an unwanted activation [6, 7] and may explain why TRF2 overexpression acts as a telomerase-independent oncogenic stimulus [8].

REGgamma proteasome activator is involved in the maintenance of chromosomal stability.

REGgamma is a member of the 11S regulatory particle that activates the 20S proteasome. Studies in REGgamma deficient mice indicated an additional role for this protein in cell cycle regulation and proliferation control. In this paper we demonstrate that REGgamma protein is equally expressed throughout the cell cycle, but undergoes a distinctive subcellular localization at mitosis. Thus, while in interphase cells REGgamma is nuclear, in telophase cells it localizes on chromosomes, suggesting a role in mitotic progression. Furthermore, we found that REGgamma overexpression weakens the mitotic arrest induced by spindle damage, allowing premature exit from mitosis, whereas REGgamma depletion has the opposite effect, thus reflecting a new REGgamma function, unrelated to its role as proteasome activator. Additionally, we found that primary cells from REGgamma-/- mice and human fibroblasts with depleted expression of REGgamma or overexpressing a dominant negative mutant unable to activate the 20S proteasome, demonstrated a marked aneuploidy (chromosomal gains and losses), supernumerary centrosomes and multipolar spindles. These findings thus underscore a previously uncharacterized function of REGgamma in centrosome and chromosomal stability maintenance.

Biochemical and cellular characterization of VRX0466617, a novel and selective inhibitor for the checkpoint kinase Chk2.

VRX0466617 is a novel selective small-molecule inhibitor for Chk2 discovered through a protein kinase screening program. In this study, we provide a detailed biochemical and cellular characterization of VRX0466617. We show that VRX0466617 blocks the enzymatic activity of recombinant Chk2, as well as the ionizing radiation (IR)-induced activation of Chk2 from cells pretreated with the compound, at doses between 5 and 10 micromol/L. These doses of VRX0466617 inhibited, to some extent, the phosphorylation of Chk2 Ser(19) and Ser(33-35), but not of Chk2 Thr(68), which is phosphorylated by the upstream ataxia-telangiectasia mutated (ATM) kinase. Interestingly, VRX0466617 induced the phosphorylation of Chk2 Thr(68) even in the absence of DNA damage, arising from the block of its enzymatic activity. VRX0466617 prevented the IR-induced Chk2-dependent degradation of Hdmx, concordant with the in vivo inhibition of Chk2. Analysis of ATM/ATM and Rad3-related substrates Smc1, p53, and Chk1 excluded a cross-inhibition of these kinases. VRX0466617 did not modify the cell cycle phase distribution, although it caused an increase in multinucleated cells. Whereas VRX0466617 attenuated IR-induced apoptosis, in short-term assays it did not affect the cytotoxicity by the anticancer drugs doxorubicin, Taxol, and cisplatin. These results underscore the specificity of VRX0466617 for Chk2, both in vitro and in vivo, and support the use of this compound as a biological probe to study the Chk2-dependent pathways.

Differential roles of ATM- and Chk2-mediated phosphorylations of Hdmx in response to DNA damage.

The p53 tumor suppressor plays a major role in maintaining genomic stability. Its activation and stabilization in response to double strand breaks (DSBs) in DNA are regulated primarily by the ATM protein kinase. ATM mediates several posttranslational modifications on p53 itself, as well as phosphorylation of p53's essential inhibitors, Hdm2 and Hdmx. Recently we showed that ATM- and Hdm2-dependent ubiquitination and subsequent degradation of Hdmx following DSB induction are mediated by phosphorylation of Hdmx on S403, S367, and S342, with S403 being targeted directly by ATM. Here we show that S367 phosphorylation is mediated by the Chk2 protein kinase, a downstream kinase of ATM. This phosphorylation, which is important for subsequent Hdmx ubiquitination and degradation, creates a binding site for 14-3-3 proteins which controls nuclear accumulation of Hdmx following DSBs. Phosphorylation of S342 also contributed to optimal 14-3-3 interaction and nuclear accumulation of Hdmx, but phosphorylation of S403 did not. Our data indicate that binding of a 14-3-3 dimer and subsequent nuclear accumulation are essential steps toward degradation of p53's inhibitor, Hdmx, in response to DNA damage. These results demonstrate a sophisticated control by ATM of a target protein, Hdmx, which itself is one of several ATM targets in the ATM-p53 axis of the DNA damage response.

DNA damage-induced cell cycle regulation and function of novel Chk2 phosphoresidues.

Chk2 kinase is activated by DNA damage to regulate cell cycle arrest, DNA repair, and apoptosis. Phosphorylation of Chk2 in vivo by ataxia telangiectasia-mutated (ATM) on threonine 68 (T68) initiates a phosphorylation cascade that promotes the full activity of Chk2. We identified three serine residues (S19, S33, and S35) on Chk2 that became phosphorylated in vivo rapidly and exclusively in response to ionizing radiation (IR)-induced DNA double-strand breaks in an ATM- and Nbs1-dependent but ataxia telangiectasia- and Rad3-related-independent manner. Phosphorylation of these residues, restricted to the G(1) phase of the cell cycle, was induced by a higher dose of IR (>1 Gy) than that required for phosphorylation of T68 (0.25 Gy) and declined by 45 to 90 min, concomitant with a rise in Chk2 autophosphorylation. Compared to the wild-type form, Chk2 with alanine substitutions at S19, S33, and S35 (Chk2(S3A)) showed impaired dimerization, defective auto- and trans-phosphorylation activities, and reduced ability to promote degradation of Hdmx, a phosphorylation target of Chk2 and regulator of p53 activity. Besides, Chk2(S3A) failed to inhibit cell growth and, in response to IR, to arrest G(1)/S progression. These findings underscore the critical roles of S19, S33, and S35 and argue that these phosphoresidues may serve to fine-tune the ATM-dependent response of Chk2 to increasing amounts of DNA damage.

Activation of ATM and Chk2 kinases in relation to the amount of DNA strand breaks.

The diverse checkpoint responses to DNA damage may reflect differential sensitivities by molecular components of the damage-signalling network to the type and amount of lesions. Here, we determined the kinetics of activation of the checkpoint kinases ATM and Chk2 (the latter substrate of ATM) in relation to the initial yield of genomic DNA single-strand (SSBs) and double-strand breaks (DSBs). We show that doses of gamma-radiation (IR) as low as 0.25 Gy, which generate vast numbers of SSBs but only a few DSBs per cell (<8), promptly activate ATM kinase and induce the phosphorylation of the ATM substrates p53-Ser15, Nbs1-Ser343 and Chk2-Thr68. The full activation of Chk2 kinase, however, is triggered by treatments inflicting >19 DSBs per cell (e.g. 1 Gy), which cause Chk2 autophosphorylation on Thr387, Chk2-dependent accumulation of p21waf1 and checkpoint arrest in the S phase. Our results indicate that, in contrast to ATM, Chk2 activity is triggered by a greater number of DSBs, implying that, below a certain threshold level of lesions (<19 DSBs), DNA repair can occur through ATM, without enforcing Chk2-dependent checkpoints.