Autophosphorylation-dependent remodeling of the DNA-dependent protein kinase catalytic subunit regulates ligation of DNA ends

Non-homologous end joining (NHEJ) is one of the primary pathways for the repair of ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) in mammalian cells. Proteins required for NHEJ include the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), Ku, XRCC4 and DNA ligase IV. Current models predict that DNA-PKcs, Ku, XRCC4 and DNA ligase IV assemble at DSBs and that the protein kinase activity of DNA-PKcs is essential for NHEJ-mediated repair of DSBs in vivo. We previously identified a cluster of autophosphorylation sites between amino acids 2609 and 2647 of DNA-PKcs. Cells expressing DNA-PKcs in which these autophosphorylation sites have been mutated to alanine are highly radiosensitive and defective in their ability to repair DSBs in the context of extrachromosomal assays. Here, we show that cells expressing DNA-PKcs with mutated autophosphorylation sites are also defective in the repair of IR-induced DSBs in the context of chromatin. Purified DNA-PKcs proteins containing serine/threonine to alanine or aspartate mutations at this cluster of autophosphorylation sites were indistinguishable from wild-type (wt) protein with respect to protein kinase activity. However, mutant DNA-PKcs proteins were defective relative to wt DNA-PKcs with respect to their ability to support T4 DNA ligase-mediated intermolecular ligation of DNA ends. We propose that autophosphorylation of DNA-PKcs at this cluster of sites is important for remodeling of DNA-PK complexes at DNA ends prior to DNA end joining.     

DNA-PKcs基因沉默抑制人乳腺上皮细胞对低剂量辐射损伤的修复

DNA-PKcs作为DNA依赖性蛋白激酶(DNA-PK)的催化亚基在DNA双链断裂(DSBs)的非同源末端重组(NHEJ)通路中起重要的作用。本实验以人乳腺上皮细胞株MCF10F为研究对象,通过siRNA技术抑制细胞内DNA-PKcs的表达,用50cGy137CS照射细胞,测定细胞生长曲线以确定细胞对低剂量辐射(LDR)的敏感性,同时检测DNA修复相关蛋白表达的变化,旨在探讨DNA依赖蛋白激酶(DNA-PKcs)基因沉默对人乳腺上皮细胞株MCF10F低剂量辐射敏感性的影响及机制。结果显示:转染特异性siRNA可使人乳腺上皮细胞(MCF10F)DNA-PKcs基因沉默,增殖受到明显的抑制;50cGyγ射线辐射可使乳腺细胞内DNA-PKcs、Ku80、ATM、P53等DNA修复相关蛋白表达增多,但DNA-PKcs基因沉默细胞(MCF10Fpk)中,这些蛋白表达显著低于对照组(MCF10Fmock)。以上结果提示,DNA-PKcs基因沉默可引起乳腺细胞对低剂量辐射敏感性增加,其原因可能与相关DNA修复蛋白表达减少有关。     

DNA-PK is activated by nucleosomes and phosphorylates H2AX within the nucleosomes in an acetylation-dependent manner

Eukaryotic DNA is organized into nucleosomes and higher order chromatin structure, which plays an important role in the regulation of many nuclear processes including DNA repair. Non-homologous end-joining, the major pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells, is mediated by a set of proteins including DNA-dependent protein kinase (DNA-PK). DNA-PK is comprised of a large catalytic subunit, DNA-PKcs, and its regulatory subunit, Ku. Current models predict that Ku binds to the ends of broken DNA and DNA-PKcs is recruited to form the active kinase complex. Here we show that DNA-PK can be activated by nucleosomes through the ability of Ku to bind to the ends of nucleosomal DNA, and that the activated DNA-PK is capable of phosphorylating H2AX within the nucleosomes. Histone acetylation has little effect on the steps of Ku binding to nucleosomes and subsequent activation of DNA-PKcs. However, acetylation largely enhances the phosphorylation of H2AX by DNA-PK, and this acetylation effect is observed when H2AX exists in the context of nucleosomes but not in a free form. These results suggest that the phosphorylation of H2AX, known to be important for DSB repair, can be regulated by acetylation and may provide a mechanistic basis on which to understand the recent observations that histone acetylation critically functions in repairing DNA DSBs.     

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.     

Sublethal doses of β-amyloid peptide abrogate DNA-dependent protein kinase activity

Accumulation of DNA damage and deficiency in DNA repair potentially contribute to the progressive neuronal loss in neurodegenerative disorders, including Alzheimer disease (AD). In multicellular eukaryotes, double strand breaks (DSBs), the most lethal form of DNA damage, are mainly repaired by the nonhomologous end joining pathway, which relies on DNA-PK complex activity. Both the presence of DSBs and a decreased end joining activity have been reported in AD brains, but the molecular player causing DNA repair dysfunction is still undetermined. β-Amyloid (Aβ), a potential proximate effector of neurotoxicity in AD, might exert cytotoxic effects by reactive oxygen species generation and oxidative stress induction, which may then cause DNA damage. Here, we show that in PC12 cells sublethal concentrations of aggregated Aβ(25-35) inhibit DNA-PK kinase activity, compromising DSB repair and sensitizing cells to nonlethal oxidative injury. The inhibition of DNA-PK activity is associated with down-regulation of the catalytic subunit DNA-PK (DNA-PKcs) protein levels, caused by oxidative stress and reversed by antioxidant treatment. Moreover, we show that sublethal doses of Aβ(1-42) oligomers enter the nucleus of PC12 cells, accumulate as insoluble oligomeric species, and reduce DNA-PK kinase activity, although in the absence of oxidative stress. Overall, these findings suggest that Aβ mediates inhibition of the DNA-PK-dependent nonhomologous end joining pathway contributing to the accumulation of DSBs that, if not efficiently repaired, may lead to the neuronal loss observed in AD.     

Defining interactions between DNA-PK and ligase IV/XRCC4

Non-homologous end joining (NHEJ) is a major pathway for the repair of DNA double-strand breaks (DSBs) in mammalian cells. DNA-dependent protein kinase (DNA-PK), ligase IV, and XRCC4 are all critical components of the NHEJ repair pathway. DNA-PK is composed of a heterodimeric DNA-binding component, Ku, and a large catalytic subunit, DNA-PKcs. Ligase IV and XRCC4 associate to form a multimeric complex that is also essential for NHEJ. DNA-PK and ligase IV/XRCC4 interact at DNA termini which results in stimulated ligase activity. Here, we define interactions between the components of these two essential complexes, DNA-PK and ligase IV/XRCC4. We find that ligase IV/XRCC4 associates with DNA-PK in a DNA-independent manner. The specific protein-protein interactions that mediate the interaction between these two complexes are further identified. Direct interactions between ligase IV and Ku as well as between XRCC4 and DNA-PKcs are shown. In contrast, binding of ligase IV to DNA-PKcs or XRCC4 to Ku is very weak or non-existent. Our data defines the specific protein pairs involved in the association of DNA-PK and ligase IV/XRCC4, and suggests a molecular mechanism for coordinating the assembly of the DNA repair complex at DNA breaks.     

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.     

Involvement of DNA-dependent protein kinase in UV-induced replication arrest.

Cells exposed to UV irradiation are predominantly arrested at S-phase as well as at the G(1)/S boundary while repair occurs. It is not known how UV irradiation induces S-phase arrest and yet permits DNA repair; however, UV-induced inhibition of replication is efficiently reversed by the addition of replication protein A (RPA), suggesting a role for RPA in this regulatory event. Here, we show evidence that DNA-dependent protein kinase (DNA-PK), plays a role in UV-induced replication arrest. DNA synthesis of M059K (DNA-PK catalytic subunit-positive (DNA-PKcs(+))), as measured by [(3)H]thymidine incorporation, was significantly arrested by 4 h following UV irradiation, whereas M059J (DNA-PKcs(-)) cells were much less affected. Similar results were obtained with the in vitro replication reactions where immediate replication arrest occurred in DNA-PKcs(+) cells following UV irradiation, and only a gradual decrease in replication activity was observed in DNA-PKcs(-) cells. Reversal of replication arrest was observed at 8 h following UV irradiation in DNA-PKcs(+) cells but not in DNA-PKcs(-) cells. Reversal of UV-induced replication arrest was also observed in vitro by the addition of a DNA-PK inhibitor, wortmannin, or by immunodepletion of DNA-PKcs, supporting a positive role for DNA-PK in damage-induced replication arrest. The RPA-containing fraction from UV-irradiated DNA-PKcs(+) cells poorly supported DNA replication, whereas the replication activity of the RPA-containing fraction from DNA-PKcs(-) cells was not affected by UV, suggesting that DNA-PKcs may be involved in UV-induced replication arrest through modulation of RPA activity. Together, our results strongly suggest a role for DNA-PK in S-phase (replication) arrest in response to UV irradiation.     

The N-terminal Region of the DNA-dependent Protein Kinase Catalytic Subunit Is Required for Its DNA Double-stranded Break-mediated Activation

DNA-dependent protein kinase (DNA-PK) plays an essential role in the repair of DNA double-stranded breaks (DSBs) mediated by the nonhomologous end-joining pathway. DNA-PK is a holoenzyme consisting of a DNA-binding (Ku70/Ku80) and catalytic (DNA-PKcs) subunit. DNA-PKcs is a serine/threonine protein kinase that is recruited to DSBs via Ku70/80 and is activated once the kinase is bound to the DSB ends. In this study, two large, distinct fragments of DNA-PKcs, consisting of the N terminus (amino acids 1–2713), termed N-PKcs, and the C terminus (amino acids 2714–4128), termed C-PKcs, were produced to determine the role of each terminal region in regulating the activity of DNA-PKcs. N-PKcs but not C-PKcs interacts with the Ku-DNA complex and is required for the ability of DNA-PKcs to localize to DSBs. C-PKcs has increased basal kinase activity compared with DNA-PKcs, suggesting that the N-terminal region of DNA-PKcs keeps basal activity low. The kinase activity of C-PKcs is not stimulated by Ku70/80 and DNA, further supporting that the N-terminal region is required for binding to the Ku-DNA complex and full activation of kinase activity. Collectively, the results show the N-terminal region mediates the interaction between DNA-PKcs and the Ku-DNA complex and is required for its DSB-induced enzymatic activity.     

Genomic sequences capable of committing mouse and rat fibroblasts to adipogenesis.

The mouse Swiss 3T3-F442A/3T3-C2 cell system is well suited for the isolation of genes involved in commitment to adipogenesis. 3T3-F442A cells convert to adipocytes with high efficiency in response to confluence and insulin. The sister clonal line 3T3-C2 does not respond to these signals, but can convert to adipocytes when transfected with DNA from 3T3-F442A preadipocytes or from human fat. Human fat-tissue biopsy FO46 DNA transfected into 3T3-C2 gave rise to fat foci after two rounds of transfection and selection. A cosmid library of a subclone of secondary transfectant 3T3-C2/FO46-1 was screened for the human repetitive Alu sequence. Five out of eight Alu+ recombinant clones committed 3T3-C2 cells to adipogenesis. The adipose commitment (AC) activity of one cosmid, p18A4, was found to reside in two small, non-identical, subcloned sequences 1.2kb and 2.0kb in length, each separately able to commit 3T3-C2, precrisis mouse and rat fibroblasts and the multipotential C3H10T1/2 cell line to adipogenesis. We conclude that commitment to adipogenesis can be effected in vitro with high efficiency by transfection of specific sequences into a variety of host cells.     

Shorter telomeres, accelerated ageing and increased lymphoma in DNA-PKcs-deficient mice

Non-homologous end joining (NHEJ) is the principal repair mechanism used by mammalian cells to cope with double-strand breaks (DSBs) that continually occur in the genome. One of the key components of the mammalian NHEJ machinery is the DNA-PK complex, formed by the Ku86/70 heterodimer and the DNA-PK catalytic subunit (DNA-PKcs). Here, we report on the detailed life-long follow-up of DNA-PKcs-defective mice. Apart from defining a role of DNA-PKcs in telomere length maintenance in the context of the ageing organism, we observed that DNA-PKcs-defective mice had a shorter life span and showed an earlier onset of ageing-related pathologies than the corresponding wild-type littermates. In addition, DNA-PKcs ablation was associated with a markedly higher incidence of T lymphomas and infections. In conclusion, these data link the dual role of DNA-PKcs in DNA repair and telomere length maintenance to organismal ageing and cancer.     

The activity of the DNA-dependent protein kinase (DNA-PK) complex is determinant in the cellular response to nitrogen mustards

The DNA-dependent protein kinase plays a critical role in mammalian DNA double strand break (DSB) repair and in specialized recombination, such as lymphoid V(D)J recombination. Its regulatory subunit Ku (dimer of the Ku70 and Ku80 protein) binds to DNA and recruits the kinase catalytic sub-unit, DNA-PKcs. We show here that three different strains deficient in either the Ku80 (xrs-6) or DNA-PKcs (V-3, scid) component of DNA-PK are markedly sensitive (3.5- to 5-fold) to a group of DNA cross-linking agents, the nitrogen mustards (NMs) (melphalan and mechlorethamine) as compared to their parental cell line. Importantly, the level of hypersensitivity to these drugs was close to the level of hypersensitivity observed for radiomimetic agents that create DSBs in DNA (bleomycin and neocarzinostatin). In addition, sensitivity to NMs was restored to the parental level in the xrs-6 cell line stably transfected with the human Ku80 gene (xrs-6/Ku80), showing unequivocally that DNA-PK is involved in this phenotype. These results indicate that a function of the whole DNA-PK protein complex is involved in the cellular response to NMs and suggest that the repair of DNA interstrand cross-links induced in DNA by NMs involved a DNA-PK dependent pathway that shares common features with DNA DSBs repair.     

Repair of a minimal DNA double-strand break by NHEJ requires DNA-PKcs and is controlled by the ATM/ATR checkpoint

Mammalian cells primarily rejoin DNA double-strand breaks (DSBs) by the non-homologous end-joining (NHEJ) pathway. The joining of the broken DNA ends appears directly without template and accuracy is ensured by the NHEJ factors that are under ATM/ATR regulated checkpoint control. In the current study we report the engineering of a mono-specific DNA damaging agent. This was used to study the molecular requirements for the repair of the least complex DSB in vivo. Single-chain PvuII restriction enzymes fused to protein delivery sequences transduce cells efficiently and induce blunt end DSBs in vivo. We demonstrate that beside XRCC4/LigaseIV and KU, the DNA-PK catalytic subunit (DNA-PKcs) is also essential for the joining of this low complex DSB in vivo. The appearance of blunt end 3′-hydroxyl and 5′-phosphate DNA DSBs induces a significantly higher frequency of anaphase bridges in cells that do not contain functional DNA-PKcs, suggesting an absolute requirement for DNA-PKcs in the control of chromosomal stability during end joining. Moreover, these minimal blunt end DSBs are sufficient to induce a p53 and ATM/ATR checkpoint function.     

DNA-dependent protein kinase catalytic subunit: a target for an ICE-like protease in apoptosis.

Radiosensitive cell lines derived from X-ray cross complementing group 5 (XRCC5), SCID mice and a human glioma cell line lack components of the DNA-dependent protein kinase, DNA-PK, suggesting that DNA-PK plays an important role in DNA double-strand break repair. Another enzyme implicated in DNA repair, poly(ADP-ribose) polymerase, is cleaved and inactivated during apoptosis, suggesting that some DNA repair proteins may be selectively targeted for destruction during apoptosis. Here we demonstrate that DNA-PKcs, the catalytic subunit of DNA-PK, is preferentially degraded after the exposure of different cell types to a variety of agents known to cause apoptosis. However, Ku, the DNA-binding component of the enzyme, remains intact. Degradation of DNA-PKcs was accompanied by loss of DNA-PK activity. One cell line resistant to etoposide-induced apoptosis failed to show degradation of DNA-PKcs. Protease inhibitor data implicated an ICE-like protease in the cleavage of DNA-PKcs, and it was subsequently shown that the cysteine protease CPP32, but not Mch2alpha, ICE or TX, cleaved purified DNA-PKcs into three fragments of comparable size with those observed in cells undergoing apoptosis. Cleavage sites in DNA-PKcs, determined by antibody mapping and microsequencing, were shown to be the same for CPP32 cleavage and for cleavage catalyzed by extracts from cells undergoing apoptosis. These observations suggest that DNA-PKcs is a critical target for proteolysis by an ICE-like protease during apoptosis.     

Function of DNA-protein kinase catalytic subunit during the early meiotic prophase without Ku70 and Ku86

All components of the double-stranded DNA break (DSB) repair complex DNA-dependent protein kinase (DNA-PK), including Ku70, Ku86, and DNA-PK catalytic subunit (DNA-PKcs), were found in the radiosensitive spermatogonia. Although p53 induction was unaffected, spermatogonial apoptosis occurred faster in the irradiated DNA-PKcs-deficient scid testis. This finding suggests that spermatogonial DNA-PK functions in DNA damage repair rather than p53 induction. Despite the fact that early spermatocytes lack the Ku proteins, spontaneous apoptosis of these cells occurred in the scid testis. The majority of these apoptotic spermatocytes were found at stage IV of the cycle of the seminiferous epithelium where a meiotic checkpoint has been suggested to exist. Meiotic synapsis and recombination during the early meiotic prophase induce DSBs, which are apparently less accurately repaired in scid spermatocytes that then fail to pass the meiotic checkpoint. The role for DNA-PKcs during the meiotic prophase differs from that in mitotic cells; it is not influenced by ionizing radiation and is independent of the Ku heterodimer.     

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.     

DNA-dependent protein kinase catalytic subunit. Structural requirements for kinase activation by DNA ends.

DNA-dependent protein kinase (DNA-PK) is a DNA end-activated protein kinase composed of a catalytic subunit, DNA-PKcs, and a DNA binding subunit, Ku, that is involved in repair of DNA double-stranded breaks (DSBs). We have previously shown that DNA-PKcs interacts with single-stranded DNA (ssDNA) ends with a separate ssDNA binding site to be activated for its kinase activity. Here, the properties of the ssDNA binding site were examined by using DNA fragments with modified ssDNA extensions. DNA fragments with a wide range of ssDNA modifictations activated DNA-PKcs, indicating a relaxed specificity for the chemical structure of terminal nucleotides of a DSB. Methyl substitution of the phosphate backbone impaired kinase activation but not binding, indicating that interaction with the DNA backbone was involved in kinase activation. Experiments with RNA and RNA/DNA hybrid fragments suggested that the discrimination between RNA and DNA ends resides in the double-stranded DNA binding function of DNA-PKcs. DNA fragments exposing only one ssDNA end activated DNA-PKcs poorly, suggesting that DNA-PKcs distinguishes between DSBs and ssDNA breaks by simultaneous interaction with two ssDNA ends. These properties potentially explain how DNA-PKcs can be specifically activated by DSBs but still recognize the diverse chemical structures exposed when DSBs are introduced by ionizing radiation.     

UV sensitivity and impaired nucleotide excision repair in DNA-dependent protein kinase mutant cells.

DNA-dependent protein kinase (DNA-PK), a member of the phosphatidyl-inositol (PI)3-kinase family, is involved in the repair of DNA double-strand breaks. Its regulatory subunit, Ku, binds to DNA and recruits the kinase catalytic subunit (DNA-PKcs). We show here a new role of DNA-PK in the modulation of the process of nucleotide excision repair (NER) in vivo since, as compared with their respective parental cell lines, DNA-PK mutants (scid , V-3 and xrs 6 cells) exhibit sensitivity to UV-C irradiation (2.0- to 2.5-fold) and cisplatin ( approximately 3- to 4-fold) associated with a decreased activity (40-55%) of unscheduled DNA synthesis after UV-C irradiation. Moreover, we observed that wortmannin sensitized parental cells in vivo when combined with either cisplatin or UV-C light, but had no effect on the DNA-PKcs deficient scid cells. Despite a lower repair synthesis activity (approximately 2-fold) measured in vitro with nuclear cell extracts from DNA-PK mutants, a direct involvement of DNA-PK in the NER reaction in vitro has not been observed. This study establishes a regulatory function of DNA-PK in the NER process in vivo but rules out a physical role of the complex in the repair machinery at the site of the DNA lesion.     

Double strand break rejoining by the Ku-dependent mechanism of non-homologous end-joining

The DNA-dependent protein kinase functions in the repair of DNA double strand breaks (DSBs) and in V(D)J recombination. To gain insight into the function of DNA-PK in this process we have carried out a mutation analysis of Ku80 and DNA-PKcs. Mutations at multiple sites within the N-terminal two thirds of Ku80 result in loss of Ku70/80 interaction, loss of DNA end-binding activity and inability to complement Ku80 defective cell lines. In contrast, mutations in the carboxy terminal region of the protein do not impair DNA end-binding activity but decrease the ability of Ku to activate DNA-PK. To gain insight into important functional domains within DNA-PKcs, we have analysed defective mutants, including the mouse scid cell line, and the rodent mutants, irs-20 and V-3. Mutational changes in the carboxy terminal region have been identified in all cases. Our results strongly suggest that the C-terminus of DNA-PKcs is required for kinase activity.     

DNA-PK autophosphorylation facilitates Artemis endonuclease activity

The Artemis nuclease is defective in radiosensitive severe combined immunodeficiency patients and is required for the repair of a subset of ionising radiation induced DNA double-strand breaks (DSBs) in an ATM and DNA-PK dependent process. Here, we show that Artemis phosphorylation by ATM and DNA-PK in vitro is primarily attributable to S503, S516 and S645 and demonstrate ATM dependent phosphorylation at serine 645 in vivo. However, analysis of multisite phosphorylation mutants of Artemis demonstrates that Artemis phosphorylation is dispensable for endonuclease activity in vitro and for DSB repair and V(D)J recombination in vivo. Importantly, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) autophosphorylation at the T2609-T2647 cluster, in the presence of Ku and target DNA, is required for Artemis-mediated endonuclease activity. Moreover, autophosphorylated DNA-PKcs stably associates with Ku-bound DNA with large single-stranded overhangs until overhang cleavage by Artemis. We propose that autophosphorylation triggers conformational changes in DNA-PK that enhance Artemis cleavage at single-strand to double-strand DNA junctions. These findings demonstrate that DNA-PK autophosphorylation regulates Artemis access to DNA ends, providing insight into the mechanism of Artemis mediated DNA end processing.