DNA-dependent protein kinase catalytic subunit is not required for dysfunctional telomere fusion and checkpoint response in the telomerase-deficient mouse

Telomeres are key structural elements for the protection and maintenance of linear chromosomes, and they function to prevent recognition of chromosomal ends as DNA double-stranded breaks. Loss of telomere capping function brought about by telomerase deficiency and gradual erosion of telomere ends or by experimental disruption of higher-order telomere structure culminates in the fusion of defective telomeres and/or the activation of DNA damage checkpoints. Previous work has implicated the nonhomologous end-joining (NHEJ) DNA repair pathway as a critical mediator of these biological processes. Here, employing the telomerase-deficient mouse model, we tested whether the NHEJ component DNA-dependent protein kinase catalytic subunit (DNA-PKcs) was required for fusion of eroded/dysfunctional telomere ends and the telomere checkpoint responses. In late-generation mTerc(-/-) DNA-PKcs(-/-) cells and tissues, chromosomal end-to-end fusions and anaphase bridges were readily evident. Notably, nullizygosity for DNA Ligase4 (Lig4)--an additional crucial NHEJ component--was also permissive for chromosome fusions in mTerc(-/-) cells, indicating that, in contrast to results seen with experimental disruption of telomere structure, telomere dysfunction in the context of gradual telomere erosion can engage additional DNA repair pathways. Furthermore, we found that DNA-PKcs deficiency does not reduce apoptosis, tissue atrophy, or p53 activation in late-generation mTerc(-/-) tissues but rather moderately exacerbates germ cell apoptosis and testicular degeneration. Thus, our studies indicate that the NHEJ components, DNA-PKcs and Lig4, are not required for fusion of critically shortened telomeric ends and that DNA-PKcs is not required for sensing and executing the telomere checkpoint response, findings consistent with the consensus view of the limited role of DNA-PKcs in DNA damage signaling in general.     

Genetic interaction between DNA polymerase beta and DNA-PKcs in embryogenesis and neurogenesis

DNA polymerase beta (Polbeta) has been implicated in base excision repair. Polbeta knockout mice exhibit apoptosis in postmitotic neuronal cells and die at birth. Also, mice deficient in nonhomologous end-joining (NHEJ), a major pathway for DNA double-strand break repair, cause massive neuronal apoptosis. Severe combined immunodeficiency (SCID) mice have a mutation in the gene encoding DNA-dependent protein kinase catalytic subunit (DNA-PKcs), the component of NHEJ, and exhibit defective lymphogenesis. To study the interaction between Polbeta and DNA-PKcs, we generated mice doubly deficient in Polbeta and DNA-PKcs. Polbeta(-/-)DNA-PKcs(scid/scid) embryos displayed greater developmental delay, more extensive neuronal apoptosis, and earlier lethality than Polbeta(-/-) and DNA-PKcs(scid/scid) embryos. Furthermore, to study the involvement of p53 in the phenotype, we generated Polbeta(-/-)DNA-PKcs(scid/scid)p53(-/-) triple-mutant mice. The mutants did not exhibit apoptosis but were lethal with defective neurulation at midgestation. These results suggest a genetic interaction between Polbeta and DNA-PKcs in embryogenesis and neurogenesis.     

Cell cycle dependence of DNA-dependent protein kinase phosphorylation in response to DNA double strand breaks

DNA-dependent protein kinase (DNA-PK), consisting of Ku and DNA-PKcs subunits, is the key component of the non-homologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair. Although the kinase activity of DNA-PKcs is essential for NHEJ, thus far, no in vivo substrate has been conclusively identified except for an autophosphorylation site on DNA-PKcs itself (threonine 2609). Here we report the ionizing radiation (IR)-induced autophosphorylation of DNA-PKcs at a novel site, serine 2056, the phosphorylation of which is required for the repair of DSBs by NHEJ. Interestingly, IR-induced DNA-PKcs autophosphorylation is regulated in a cell cycle-dependent manner with attenuated phosphorylation in the S phase. In contrast, DNA replication-associated DSBs resulted in DNA-PKcs autophosphorylation and localization to DNA damage sites. These results indicate that although IR-induced DNA-PKcs phosphorylation is attenuated in the S phase, DNA-PKcs is preferentially activated by the physiologically relevant DNA replication-associated DSBs at the sites of DNA synthesis.     

Ser18 and Ser23 Phosphorylation Plays Synergistic Roles in Activating p53-Dependent Neuronal Apoptosis

Tumor suppressor p53 is required for the neuronal apoptosis in response to DNA double-stranded break (DSB) damage. Posttranslational modifications such as phosphorylation play important roles in activating p53-dependent apoptosis after DNA damage. In support of this notion, our recent studies indicate that Ser18 and Ser23 phosphorylation together plays critical roles in activating p53 apoptotic activities in vivo. Thymocytes derived from p53S18/23A mice are essentially resistant to p53-dependent apoptosis after DNA DSB damage. In addition, identical to p53-deficiency, p53S18/23A knock-in mutation completely rescues the embryonic lethality of XRCC4-/- mice, which die of the massive p53-dependent apoptosis of embryonic neurons likely as a result of accumulated endogenous DNA damage. To dissect the contribution of Ser18 and Ser23 phosphorylation to p53-dependent neuronal apoptosis, we report here that neither p53S18A nor p53S23A mutation alone can rescue the embryonic lethality of XRCC4-/- mice. Therefore, Ser18 and Ser23 phosphorylation plays synergistic and critical roles in activating p53-dependent neuronal apoptosis.     

BRCA1 modulates the autophosphorylation status of DNA-PKcs in S phase of the cell cycle

Non-homologous end-joining (NHEJ) and homologous recombination (HR) are the two prominent pathways responsible for the repair of DNA double-strand breaks (DSBs). NHEJ is not restricted to a cell-cycle stage, whereas HR is active primarily in the S/G2 phases suggesting there are cell cycle-specific mechanisms that play a role in the choice between NHEJ and HR. Here we show NHEJ is attenuated in S phase via modulation of the autophosphorylation status of the NHEJ factor DNA-PKcs at serine 2056 by the pro-HR factor BRCA1. BRCA1 interacts with DNA-PKcs in a cell cycle-regulated manner and this interaction is mediated by the tandem BRCT domain of BRCA1, but surprisingly in a phospho-independent manner. BRCA1 attenuates DNA-PKcs autophosphorylation via directly blocking the ability of DNA-PKcs to autophosphorylate. Subsequently, blocking autophosphorylation of DNA-PKcs at the serine 2056 phosphorylation cluster promotes HR-required DNA end processing and loading of HR factors to DSBs and is a possible mechanism by which BRCA1 promotes HR.     

Akt promotes post-irradiation survival of human tumor cells through initiation, progression, and termination of DNA-PKcs-dependent DNA double-strand break repair

Akt phosphorylation has previously been described to be involved in mediating DNA damage repair through the nonhomologous end-joining (NHEJ) repair pathway. Yet the mechanism how Akt stimulates DNA-protein kinase catalytic subunit (DNA-PKcs)-dependent DNA double-strand break (DNA-DSB) repair has not been described so far. In the present study, we investigated the mechanism by which Akt can interact with DNA-PKcs and promote its function during the NHEJ repair process. The results obtained indicate a prominent role of Akt, especially Akt1 in the regulation of NHEJ mechanism for DNA-DSB repair. As shown by pull-down assay of DNA-PKcs, Akt1 through its C-terminal domain interacts with DNA-PKcs. After exposure of cells to ionizing radiation (IR), Akt1 and DNA-PKcs form a functional complex in a first initiating step of DNA-DSB repair. Thereafter, Akt plays a pivotal role in the recruitment of AKT1/DNA-PKcs complex to DNA duplex ends marked by Ku dimers. Moreover, in the formed complex, Akt1 promotes DNA-PKcs kinase activity, which is the necessary step for progression of DNA-DSB repair. Akt1-dependent DNA-PKcs kinase activity stimulates autophosphorylation of DNA-PKcs at S2056 that is needed for efficient DNA-DSB repair and the release of DNA-PKcs from the damage site. Thus, targeting of Akt results in radiosensitization of DNA-PKcs and Ku80 expressing, but not of cells deficient for, either of these proteins. The data showed indicate for the first time that Akt through an immediate complex formation with DNA-PKcs can stimulate the accumulation of DNA-PKcs at DNA-DSBs and promote DNA-PKcs activity for efficient NHEJ DNA-DSB repair.     

The role of RecQ helicases in non-homologous end-joining

Abstract

DNA double-strand breaks are highly toxic DNA lesions that cause genomic instability, if not efficiently repaired. RecQ helicases are a family of highly conserved proteins that maintain genomic stability through their important roles in several DNA repair pathways, including DNA double-strand break repair. Double-strand breaks can be repaired by homologous recombination (HR) using sister chromatids as templates to facilitate precise DNA repair, or by an HR-independent mechanism known as non-homologous end-joining (NHEJ) (error-prone). NHEJ is a non-templated DNA repair process, in which DNA termini are directly ligated. Canonical NHEJ requires DNA-PKcs and Ku70/80, while alternative NHEJ pathways are DNA-PKcs and Ku70/80 independent. This review discusses the role of RecQ helicases in NHEJ, alternative (or back-up) NHEJ (B-NHEJ) and microhomology-mediated end-joining (MMEJ) in V(D)J recombination, class switch recombination and telomere maintenance.     

DNA-PKcs-dependent signaling of DNA damage in Dictyostelium discoideum

DNA double-strand breaks (DSBs) can be repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). In vertebrates, the first step in NHEJ is recruitment of the DNA-dependent protein kinase (DNA-PK) to DNA termini. DNA-PK consists of a catalytic subunit (DNA-PKcs) that is recruited to DNA ends by the Ku70/Ku80 heterodimer. Although Ku has been identified in a wide variety of organisms, to date DNA-PKcs has only been identified experimentally in vertebrates. Here, we report the identification of DNA-PK in the nonvertebrate Dictyostelium. Dictyostelium Ku80 contains a conserved domain previously implicated in recruiting DNA-PKcs to DNA and consistent with this observation, we have identified DNA-PKcs in the Dictyostelium genome. Disruption of the gene encoding Dictyostelium DNA-PKcs results in sensitivity to DNA DSBs and defective H2AX phosphorylation in response to this form of DNA damage. However, these phenotypes are only apparent when DNA damage is administered in G(1) phase of the cell cycle. These data illustrate a cell cycle-dependent requirement for Dictyostelium DNA-PK in signaling and combating DNA DSBs and represent the first experimental verification of DNA-PKcs in a nonvertebrate organism.     

Genetic evidence for involvement of two distinct nonhomologous end-joining pathways in repair of topoisomerase II-mediated DNA damage

In vertebrate cells, DNA double-strand breaks are efficiently repaired by homologous recombination or nonhomologous end-joining (NHEJ). The latter pathway relies on Ku (the Ku70/Ku86 heterodimer), DNA-PKcs, Artemis, Xrcc4, and DNA ligase IV (Lig4). Here, we show that a human pre-B cell line nullizygous for Lig4 exhibits hypersensitivity to topoisomerase II (Top2) inhibitors, demonstrating a crucial role for the NHEJ pathway in repair of Top2-induced DNA damage in vertebrates. We also show that in the chicken DT40 cell line, all NHEJ mutants (i.e., Ku70-, Lig4-, and DNA-PKcs-null cells) are equally hypersensitive to the Top2 inhibitor ICRF-193, indicating that the drug-induced damage is repaired by NHEJ involving DNA-PKcs. Intriguingly, however, DNA-PKcs-null cells display considerably less severe phenotype than other NHEJ mutants in terms of hypersensitivity to VP-16, a Top2 poison that stabilizes cleavable complexes. The results indicate that two distinct NHEJ pathways, involving or not involving DNA-PKcs, are important for the repair of VP-16-induced DNA damage, providing additional evidence for the biological relevance of DNA-PKcs-independent NHEJ. Our results provide significant insights into the mechanisms of repair of Top2-mediated DNA damage, with implications for chemotherapy involving Top2 inhibitors.     

DNA ligase IV deficiency in mice leads to defective neurogenesis and embryonic lethality via the p53 pathway

DNA ligase IV (LIG4) is a nonhomologous end-joining (NHEJ) protein used for V(D)J recombination and DNA repair. In mice, Lig4 deficiency causes embryonic lethality, massive neuronal apoptosis, arrested lymphogenesis, and various cellular defects. Herein, we assess potential roles in this phenotype for INK4a/ARF and p53, two proteins implicated in apoptosis and senescence. INK4a/ARF deficiency rescued proliferation/senescence defects of Lig4-deficient fibroblasts but not other phenotypic aspects. In contrast, p53 deficiency rescued embryonic lethality, neuronal apoptosis, and fibroblast proliferation/senescence defects but not lymphocyte development or radiosensitivity. Young Lig4/p53 double null mice routinely died from pro-B lymphomas. Thus, in the context of Lig4 deficiency, embryonic lethality and neuronal apoptosis likely result from a p53-dependent response to unrepaired DNA damage, and neuronal apoptosis and lymphocyte developmental defects can be mechanistically dissociated.     

Visualization of DNA-induced conformational changes in the DNA repair kinase DNA-PKcs

The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is essential for the repair of double-stranded DNA breaks (DSBs) in non- homologous end joining (NHEJ) and during V(D)J recombination. DNA-PKcs binds single- and double-stranded DNA in vitro, and in vivo the Ku heterodimer probably helps recruit it to DSBs with high affinity. Once loaded onto DNA, DNA-PKcs acts as a scaffold for other repair factors to generate a multiprotein complex that brings the two DNA ends together. Human DNA-PKcs has been analysed by electron microscopy in the absence and presence of double-stranded DNA, and the three-dimensional reconstruction of DNA-bound DNA-PKcs displays large conformational changes when compared with the unbound protein. DNA-PKcs seems to use a palm-like domain to clip onto the DNA, and this new conformation correlates with the activation of the kinase. We suggest that the observed domain movements might help the binding and maintenance of DNA-PKcs' interaction with DNA at the sites of damage, and that these conformational changes activate the kinase.     

Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes

Replication protein A (RPA) is a DNA single-strand binding protein essential for DNA replication, recombination and repair. In human cells treated with the topoisomerase inhibitors camptothecin or etoposide (VP-16), we find that RPA2, the middle-sized subunit of RPA, becomes rapidly phosphorylated. This response appears to be due to DNA-dependent protein kinase (DNA-PK) and to be independent of p53 or the ataxia telangiectasia mutated (ATM) protein. RPA2 phosphorylation in response to camptothecin required ongoing DNA replication. Camptothecin itself partially inhibited DNA synthesis, and this inhibition followed the same kinetics as DNA-PK activation and RPA2 phosphorylation. DNA-PK activation and RPA2 phosphorylation were prevented by the cell-cycle checkpoint abrogator 7-hydroxystaurosporine (UCN-01), which markedly potentiates camptothecin cytotoxicity. The DNA-PK catalytic subunit (DNA-PKcs) was found to bind RPA which was replaced by the Ku autoantigen upon camptothecin treatment. DNA-PKcs interacted directly with RPA1 in vitro. We propose that the encounter of a replication fork with a topoisomerase-DNA cleavage complex could lead to a juxtaposition of replication fork-associated RPA and DNA double-strand end-associated DNA-PK, leading to RPA2 phosphorylation which may signal the presence of DNA damage to an S-phase checkpoint mechanism. Keywords: camptothecin/DNA damage/DNA-dependent protein kinase/RPA2 phosphorylation     

53BP1 deficiency combined with telomere dysfunction activates ATR-dependent DNA damage response

TRF1 protects mammalian telomeres from fusion and fragility. Depletion of TRF1 leads to telomere fusions as well as accumulation of γ-H2AX foci and activation of both the ataxia telangiectasia mutated (ATM)- and the ataxia telangiectasia and Rad3 related (ATR)-mediated deoxyribonucleic acid (DNA) damage response (DDR) pathways. 53BP1, which is also present at dysfunctional telomeres, is a target of ATM that accumulates at DNA double-strand breaks and favors nonhomologous end-joining (NHEJ) repair over ATM-dependent resection and homology-directed repair (homologous recombination [HR]). To address the role of 53BP1 at dysfunctional telomeres, we generated mice lacking TRF1 and 53BP1. 53BP1 deficiency significantly rescued telomere fusions in mouse embryonic fibroblasts (MEFs) lacking TRF1, but they showed evidence of a switch from the NHEJ- to HR-mediated repair of uncapped telomeres. Concomitantly, double-mutant MEFs showed evidence of hyperactivation of the ATR-dependent DDR. In intact mice, combined 53BP1/TRF1 deficiency in stratified epithelia resulted in earlier onset of DNA damage and increased CHK1 phosphorylation during embryonic development, leading to aggravation of skin phenotypes.     

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.     

Hyperactivation of ATM upon DNA-PKcs inhibition modulates p53 dynamics and cell fate in response to DNA damage

A functional DNA damage response is essential for maintaining genome integrity in the presence of DNA double-strand breaks. It is mainly coordinated by the kinases ATM, ATR, and DNA-PKcs, which control the repair of broken DNA strands and relay the damage signal to the tumor suppressor p53 to induce cell cycle arrest, apoptosis, or senescence. Although many functions of the individual kinases have been identified, it remains unclear how they act in concert to ensure faithful processing of the damage signal. Using specific inhibitors and quantitative analysis at the single-cell level, we systematically characterize the contribution of each kinase for regulating p53 activity. Our results reveal a new regulatory interplay in which loss of DNA-PKcs function leads to hyperactivation of ATM and amplification of the p53 response, sensitizing cells for damage-induced senescence. This interplay determines the outcome of treatment regimens combining irradiation with DNA-PKcs inhibitors in a p53-dependent manner.