Dynamics of the PI3K-like protein kinase members ATM and DNA-PKcs at DNA double strand breaks

The protein kinases ATM and DNA-PKcs play critical roles in the cellular response to DNA double strand breaks (DSBs). ATM and DNA-PKcs are activated in response to DSBs and play several important roles in propagation of the damage signal and for the repair of DNA damage. Recent work from several groups, including ours, has focused on studying the dynamics of each of these proteins at DSBs and the requirements and factors which play a role(s) in this process. The use of live cell imaging of fluorescently-tagged ATM and DNA-PKcs has allowed us to study the real-time response of these proteins to laser-generated DNA damage in vivo. Here, we will extensively discuss the behavior of the ATM and DNA-PKcs proteins at DSB sites.     

53BP1, an activator of ATM in response to DNA damage

p53 Binding protein 1 (53BP1) belongs to a family of evolutionarily conserved DNA damage checkpoint proteins with C-terminal BRCT domains and is most likely the human ortholog of the budding yeast Rad9 protein, the first cell cycle checkpoint protein to be described. 53BP1 localizes rapidly to sites of DNA double strand breaks (DSBs) and its initial recruitment to these sites has not been shown to be dependent on any other protein. Initially, 53BP1 was thought to be a mediator of DNA DSB signaling, but now it has been shown to function upstream of ataxia-telangiectasia mutated (ATM), in one of at least two parallel pathways leading to ATM activation in response to DNA damage. Currently, only a single tudor and two BRCT domains are recognized in 53BP1; however, their precise functional role is not understood. Elucidating the function of 53BP1 will be critical to understanding how cells recognize DNA DSBs and how ATM is activated.     

Activation of ataxia telangiectasia mutated by DNA strand break-inducing agents correlates closely with the number of DNA double strand breaks

The protein kinase ataxia telangiectasia mutated (ATM) is activated when cells are exposed to ionizing radiation (IR). It has been assumed that ATM is specifically activated by the few induced DNA double strand breaks (DSBs), although little direct evidence for this assumption has been presented. DSBs constitute only a few percent of the IR-induced DNA damage, whereas the more frequent single strand DNA breaks (SSBs) and base damage account for over 98% of the overall DNA damage. It is therefore unclear whether DSBs are the only IR-induced DNA lesions that activate ATM. To test directly whether or not DSBs are responsible for ATM activation, we exposed cells to drugs and radiation that produce different numbers of DSBs and SSBs. We determined the resulting ATM activation by measuring the amount of phosphorylated Chk2 and the numbers of SSBs and DSBs in the same cells after short incubation periods. We found a strong correlation between the number of DSBs and ATM activation but no correlation with the number of SSBs. In fact, hydrogen peroxide, which, similar to IR, induces DNA damage through hydroxyl radicals but fails to induce DSBs, did not activate ATM. In contrast, we found that calicheamicin-induced strand breaks activated ATM more efficiently than IR and that ATM activation correlated with the relative DSB induction by these agents. Our data indicate that ATM is specifically activated by IR-induced DSBs, with little or no contribution from SSBs and other types of DNA damage. These findings have implications for how ATM might recognize DSBs in cells.     

The radiomimetic enediyne C-1027 induces unusual DNA damage responses to double-strand breaks

Cells lacking the protein kinase ataxia telangiectasia mutated (ATM) have defective responses to DNA double-strand breaks (DSBs), including an inability to activate damage response proteins such as p53. However, we previously showed that cells lacking ATM robustly activate p53 in response to DNA strand breaks induced by the radiomimetic enediyne C-1027. To gain insight into the nature of C-1027-induced ATM-independent damage responses to DNA DSBs, we further examined the molecular mechanisms underlying the cellular response to this unique radiomimetic agent. Like ionizing radiation (IR) and other radiomimetics, breaks induced by C-1027 efficiently activate ATM by phosphorylation at Ser1981, yet unlike other radiomimetics and IR, DNA breaks induced by C-1027 result in normal phosphorylation of p53 and the cell cycle checkpoint kinases (Chk1 and Chk2) in the absence of ATM. In the presence of ATM, but under ATM and Rad3-related kinase (ATR) deficient conditions, C-1027 treatment resulted in a decrease in the level of Chk1 phosphorylation but not in the level of p53 and Chk2 phosphorylation. Only when cells were deficient in both ATM and ATR was there a reduction in the level of phosphorylation of each of these DNA damage response proteins. This reduction was also accompanied by an increased level of cell death in comparison to that of wild-type cells or cells lacking either ATM or ATR. Our findings demonstrate a unique cellular response to C-1027-induced DNA DSBs in that DNA damage response proteins are unaffected by the absence of ATM, as long as ATR is present.     

Regulation of human polλ by ATM-mediated phosphorylation during non-homologous end joining

DNA double strand breaks (DSBs) trigger a variety of cellular signaling processes, collectively termed the DNA-damage response (DDR), that are primarily regulated by protein kinase ataxia-telangiectasia mutated (ATM). Among DDR activated processes, the repair of DSBs by non-homologous end joining (NHEJ) is essential. The proper coordination of NHEJ factors is mainly achieved through phosphorylation by an ATM-related kinase, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the molecular basis for this regulation has yet to be fully elucidated. In this study we identify the major NHEJ DNA polymerase, DNA polymerase lambda (Polλ), as a target for both ATM and DNA-PKcs in human cells. We show that Polλ is efficiently phosphorylated by DNA-PKcs in vitro and predominantly by ATM after DSB induction with ionizing radiation (IR) in vivo. We identify threonine 204 (T204) as a main target for ATM/DNA-PKcs phosphorylation on human Polλ, and establish that its phosphorylation may facilitate the repair of a subset of IR-induced DSBs and the efficient Polλ-mediated gap-filling during NHEJ. Molecular evidence suggests that Polλ phosphorylation might favor Polλ interaction with the DNA-PK complex at DSBs. Altogether, our work provides the first demonstration of how Polλ is regulated by phosphorylation to connect with the NHEJ core machinery during DSB repair in human cells.     

"ATR activation in response to ionizing radiation: still ATM territory"

ABSTRACT : Unrepaired DNA double-strand breaks (DSBs) are a major cause for genomic instability. Therefore, upon detection of a DSB a rapid response must be assembled to coordinate the proper repair/signaling of the lesion or the elimination of cells with unsustainable amounts of DNA damage. Three members of the PIKK family of protein kinases -ATM, ATR and DNA-PKcs- take the lead and initiate the signaling cascade emanating from DSB sites. Whereas DNA-PKcs activity seems to be restricted to the phosphorylation of targets involved in DNA repair, ATM and ATR phosphorylate a broad spectrum of cell cycle regulators and DNA repair proteins. In the canonical model, ATM and ATR are activated by two different types of lesions and signal through two independent and alternate pathways. Specifically, ATR is activated by various forms of DNA damage, including DSBs, arising at stalled replication forks ("replication stress"), and ATM is responsible for the signaling of DSBs that are not associated with the replication machinery throughout the cell cycle. Recent evidence suggests that this model might be oversimplified and that coordinated crosstalk between ATM and ATR activation routes goes on at the core of the DNA damage response.     

Drosophila ATM and ATR have distinct activities in the regulation of meiotic DNA damage and repair

Ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia-related (ATR) kinases are conserved regulators of cellular responses to double strand breaks (DSBs). During meiosis, however, the functions of these kinases in DSB repair and the deoxyribonucleic acid (DNA) damage checkpoint are unclear. In this paper, we show that ATM and ATR have unique roles in the repair of meiotic DSBs in Drosophila melanogaster. ATR mutant analysis indicated that it is required for checkpoint activity, whereas ATM may not be. Both kinases phosphorylate H2AV (γ-H2AV), and, using this as a reporter for ATM/ATR activity, we found that the DSB repair response is surprisingly dynamic at the site of DNA damage. γ-H2AV is continuously exchanged, requiring new phosphorylation at the break site until repair is completed. However, most surprising is that the number of γ-H2AV foci is dramatically increased in the absence of ATM, but not ATR, suggesting that the number of DSBs is increased. Thus, we conclude that ATM is primarily required for the meiotic DSB repair response, which includes functions in DNA damage repair and negative feedback control over the level of programmed DSBs during meiosis.     

A Functional Link Between SIRT1 Deacetylase and NBS1 in DNA Damage Response

In mammalian cells, DNA is often subjected to stresses such as ionizing radiation (IR) and ultraviolet light that can induce DNA double strand breaks (DSBs). In response to DNA DSBs, mammalian cells activate a rapid phosphorylation signaling cascade through the protein kinases, Ataxia-Telangiectasia Mutated (ATM) and ATM- and Rad3-Related (ATR).1 Many well-characterized DNA repair factors are phosphorylated by ATM in response to DSBs, and the sequential phosphorylation of some of these factors, including NBS1, delay cell cycle progression (checkpoint arrest) to allow time for DNA damage repair.2 Results from a new study suggest that phosphorylation of NBS1 is regulated by the acetylation status of the protein, which is modulated by SIRT1 deacetylase.     

The histone chaperone ASF1 regulates the activation of ATM and DNA-PKcs in response to DNA double-strand breaks

The Ataxia-telangiectasia mutated (ATM) kinase and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are activated by DNA double-strand breaks (DSBs). These DSBs occur in the context of chromatin but how chromatin influences the activation of these kinases is not known. Here we show that loss of the replication-dependent chromatin assembly factors ASF1A/B or CAF-1 compromises ATM activation, while augmenting DNA-PKcs activation, in response to DNA DSBs. Cells deficient in ASF1A/B or CAF-1 exhibit reduced histone H4 lysine 16 acetylation (H4K16ac), a histone mark known to promote ATM activation. ASF1A interacts with the histone acetyl transferase, hMOF that mediates H4K16ac. ASF1A depletion leads to increased recruitment of DNA-PKcs to DSBs. We propose normal chromatin assembly and H4K16ac during DNA replication is required to regulate ATM and DNA-PKcs activity in response to the subsequent induction of DNA DSBs.     

Involvement of Matrin 3 and SFPQ/NONO in the DNA damage response

The DNA damage response (DDR) is a complex signaling network that is induced by DNA lesions and vigorously activated by double strand breaks (DSBs). The DSB response is mobilized by the nuclear protein kinase ATM, which phosphorylates key players in its various branches. SFPQ (PSF) and NONO (p54) are nuclear proteins that interact with each other and have diverse roles in nucleic acids metabolism. The SFPQ/NONO heterodimer was previously found to enhance DNA strand break rejoining in vitro. Our attention was drawn to these two proteins as they interact with the nuclear matrix protein Matrin 3 (MATR3), which we found to be a novel ATM target. We asked whether SFPQ and NONO too are involved in the DSB response. Proteins that function at the early phase of this response are often recruited to the damaged sites. We observed rapid recruitment of SFPQ/NONO to sites of DNA damage induced by laser microbeam. In MATR3 knockdown cells SFPQ/NONO retention at DNA damage sites was prolonged. SFPQ and MATR3 depletion led to abnormal accumulation of cells at the S-phase of the cell cycle following treatment with the radiomimetic chemical neocarzinostatin. Notably, proteins involved in DSB repair via nonhomologous end-joining co-immunoprecipitated with NONO; SFPQ depletion delayed DSB repair. Collectively the data suggest that SFPQ, NONO and MATR3 are involved in the early stage of the DSB response, setting the scene for DSB repair.     

Repair of chromosomal RAG-mediated DNA breaks by mutant RAG proteins lacking phosphatidylinositol 3-like kinase consensus phosphorylation sites

Ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunits (DNA-PKcs) are members of the phosphatidylinositol 3-like family of serine/threonine kinases that phosphorylate serines or threonines when positioned adjacent to a glutamine residue (SQ/TQ). Both kinases are activated rapidly by DNA double-strand breaks (DSBs) and regulate the function of proteins involved in DNA damage responses. In developing lymphocytes, DSBs are generated during V(D)J recombination, which is required to assemble the second exon of all Ag receptor genes. This reaction is initiated through a DNA cleavage step by the RAG1 and RAG2 proteins, which together comprise an endonuclease that generates DSBs at the border of two recombining gene segments and their flanking recombination signals. This DNA cleavage step is followed by a joining step, during which pairs of DNA coding and signal ends are ligated to form a coding joint and a signal joint, respectively. ATM and DNA-PKcs are integrally involved in the repair of both signal and coding ends, but the targets of these kinases involved in the repair process have not been fully elucidated. In this regard, the RAG1 and RAG2 proteins, which each have several SQ/TQ motifs, have been implicated in the repair of RAG-mediated DSBs. In this study, we use a previously developed approach for studying chromosomal V(D)J recombination that has been modified to allow for the analysis of RAG1 and RAG2 function. We show that phosphorylation of RAG1 or RAG2 by ATM or DNA-PKcs at SQ/TQ consensus sites is dispensable for the joining step of V(D)J recombination.     

Breadth by depth: expanding our understanding of the repair of transposon-induced DNA double strand breaks via deep-sequencing

The transposases of DNA transposable elements catalyze the excision of the element from the host genome, but are not involved in the repair of the resulting double-strand break. To elucidate the role of various host DNA repair and damage response proteins in the repair of the hairpin-ended double strand breaks (DSBs) generated during excision of the maize Ac element in Arabidopsis thaliana, we deep-sequenced hundreds of thousands of somatic excision products from a variety of repair- or response-defective mutants. We find that each of these repair/response defects negatively affects the preservation of the ends, resulting in an enhanced frequency of deletions, insertions, and inversions at the excision site. The spectra of the resulting repair products demonstrate, not unexpectedly, that the canonical nonhomologous end joining (NHEJ) proteins DNA ligase IV and KU70 play an important role in the repair of the lesion generated by Ac excision. Our data also indicate that auxiliary NHEJ repair proteins such as DNA ligase VI and DNA polymerase lambda are routinely involved in the repair of these lesions. Roles for the damage response kinases ATM and ATR in the repair of transposition-induced DSBs are also discussed.     

Regulation of DNA double-strand break repair pathway choice

DNA double-strand breaks （DSBs） are critical lesions that can result in cell death or a wide variety of genetic alterations including largeor small-scale deletions, loss of heterozygosity, translocations, and chromosome loss. DSBs are repaired by non-homologous end-joining （NHEJ） and homologous recombination （HR）, and defects in these pathways cause genome instability and promote tumorigenesis. DSBs arise from endogenous sources including reactive oxygen species generated during cellular metabolism, collapsed replication forks, and nucleases, and from exogenous sources including ionizing radiation and chemicals that directly or indirectly damage DNA and are commonly used in cancer therapy. The DSB repair pathways appear to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type. Here we review the regulatory factors that regulate DSB repair by NHEJ and HR in yeast and higher eukaryotes. These factors include regulated expression and phosphorylation of repair proteins, chromatin modulation of repair factor accessibility, and the availability of homologous repair templates. While most DSB repair proteins appear to function exclusively in NHEJ or HR, a number of proteins influence both pathways, including the MRE11/RAD50/NBS1（XRS2） complex, BRCA1, histone H2AX, PARP-1, RAD18, DNA-dependent protein kinase catalytic subunit （DNA-PKcs）, and ATM. DNA-PKcs plays a role in mammalian NHEJ, but it also influences HR through a complex regulatory network that may involve crosstalk with ATM, and the regulation of at least 12 proteins involved in HR that are phosphorylated by DNA-PKcs and/or ATM.     

Endogenously induced DNA double strand breaks arise in heterochromatic DNA regions and require ataxia telangiectasia mutated and Artemis for their repair

Ataxia telangiectasia (ATM) mutated and Artemis, the proteins defective in ataxia telangiectasia and a class of Radiosensitive-Severe Combined Immunodeficiency (RS-SCID), respectively, function in the repair of DNA double strand breaks (DSBs), which arise in heterochromatic DNA (HC-DSBs) following exposure to ionizing radiation (IR). Here, we examine whether they have protective roles against oxidative damage induced and/or endogenously induced DSBs. We show that DSBs generated following acute exposure of G0/G1 cells to the oxidative damaging agent, tert-butyl hydroperoxide (TBH), are repaired with fast and slow components of similar magnitude to IR-induced DSBs and have a similar requirement for ATM and Artemis. Strikingly, DSBs accumulate in ATM(-/-) mouse embryo fibroblasts (MEFs) and in ATM or Artemis-defective human primary fibroblasts maintained for prolonged periods under confluence arrest. The accumulated DSBs localize to HC-DNA regions. Collectively, the results provide strong evidence that oxidatively induced DSBs arise in HC as well as euchromatic DNA and that Artemis and ATM function in their repair. Additionally, we show that Artemis functions downstream of ATM and is dispensable for HC-relaxation and for pKAP-1 foci formation. These findings are important for evaluating the impact of endogenously arising DNA DSBs in ATM and Artemis-deficient patients.     

DNA-PK and ATM phosphorylation sites in XLF/Cernunnos are not required for repair of DNA double strand breaks

Nonhomologous end joining (NHEJ) is the major pathway for the repair of DNA double strand breaks (DSBs) in human cells. NHEJ requires the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), Ku70, Ku80, XRCC4, DNA ligase IV and Artemis, as well as DNA polymerases mu and lambda and polynucleotide kinase. Recent studies have identified an additional participant, XLF, for XRCC4-like factor (also called Cernunnos), which interacts with the XRCC4-DNA ligase IV complex and stimulates its activity in vitro, however, its precise role in the DNA damage response is not fully understood. Since the protein kinase activity of DNA-PKcs is required for NHEJ, we asked whether XLF might be a physiological target of DNA-PK. Here, we have identified two major in vitro DNA-PK phosphorylation sites in the C-terminal region of XLF, serines 245 and 251. We show that these represent the major phosphorylation sites in XLF in vivo and that serine 245 is phosphorylated in vivo by DNA-PK, while serine 251 is phosphorylated by Ataxia-Telangiectasia Mutated (ATM). However, phosphorylation of XLF did not have a significant effect on the ability of XLF to interact with DNA in vitro or its recruitment to laser-induced DSBs in vivo. Similarly, XLF in which the identified in vivo phosphorylation sites were mutated to alanine was able to complement the DSB repair defect as well as radiation sensitivity in XLF-deficient 2BN cells. We conclude that phosphorylation of XLF at these sites does not play a major role in the repair of IR-induced DSBs in vivo.     

Responding to chromosomal breakage during M-phase: insights from a cell-free system

DNA double strand breaks (DSBs) activate ATM and ATR dependent checkpoints that prevent the onset of mitosis. However, how cells react to DSBs occurring when they are already in mitosis is poorly understood. The Xenopus egg extract has been utilized to study cell cycle progression and DNA damage checkpoints. Recently this system has been successfully used to uncover an ATM and ATR dependent checkpoint affecting centrosome driven spindle assembly. These studies have led to the identification of XCEP63 as major target of this pathway. XCEP63 is a coiled-coil rich protein localized at centrosome essential for proper spindle assembly. ATM and ATR directly phosphorylate XCEP63 on serine 560 inducing its delocalization from centrosome, which in turn delays spindle assembly. This pathway might contribute to regulate DNA repair or mitotic cell survival in the presence of chromosome breakage.     

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.     

Scanning fluorescence correlation spectroscopy techniques to quantify the kinetics of DNA double strand break repair proteins after γ-irradiation and bleomycin treatment

A common feature of DNA repair proteins is their mobilization in response to DNA damage. The ability to visualizing and quantifying the kinetics of proteins localizing/dissociating from DNA double strand breaks (DSBs) via immunofluorescence or live cell fluorescence microscopy have been powerful tools in allowing insight into the DNA damage response, but these tools have some limitations. For example, a number of well-established DSB repair factors, in particular those required for non-homologous end joining (NHEJ), do not form discrete foci in response to DSBs induced by ionizing radiation (IR) or radiomimetic drugs, including bleomycin, in living cells. In this report, we show that time-dependent kinetics of the NHEJ factors Ku80 and DNA-dependent protein kinase catalytic subunits (DNA–PKcs) in response to IR and bleomycin can be quantified by Number and Brightness analysis and Raster-scan Image Correlation Spectroscopy. Fluorescent-tagged Ku80 and DNA–PKcs quickly mobilized in response to IR and bleomycin treatments consistent with prior reports using laser-generated DSBs. The response was linearly dependent on IR dose, and blocking NHEJ enhanced immobilization of both Ku80 and DNA–PKcs after DNA damage. These findings support the idea of using Number and Brightness and Raster-scan Image Correlation Spectroscopy as methods to monitor kinetics of DSB repair proteins in living cells under conditions mimicking radiation and chemotherapy treatments.