• Users Online: 291
  • Home
  • Print this page
  • Email this page
Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contacts Login 

 Table of Contents  
Year : 2020  |  Volume : 11  |  Issue : 4  |  Page : 123-134

DNA-Dependent protein kinase in DNA damage response: Three decades and beyond

1 Laboratory for Advanced Nuclear Energy, Institute of Innovative Research, Tokyo Institute of Technology, Tokyo, Japan
2 Laboratory for Advanced Nuclear Energy, Institute of Innovative Research, Tokyo Institute of Technology, Tokyo, Japan; Department of Zoology, SPC Government College, Ajmer, Rajasthan, India

Date of Submission11-Nov-2020
Date of Acceptance11-Dec-2020
Date of Web Publication30-Dec-2020

Correspondence Address:
Dr. Yoshihisa Matsumoto
Laboratory for Advanced Nuclear Energy, Institute of Innovative Research, Tokyo Institute of Technology, Tokyo
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jrcr.jrcr_60_20

Rights and Permissions

Ionizing radiation exerts various biological effects, including cell killing and carcinogenesis, mainly through generating damage on DNA. Among various types of DNA damage, DNA double-strand break (DSB) is considered the most deleterious and most intimately related to biolog?ical effects of radiation. DNA-dependent protein kinase (DNA-PK), consisting of DNA-PK catalytic subunit and Ku80-Ku70 heterodimer (Ku), is activated upon binding to the end of double-stranded DNA and acts as the molecular sensor for DSB. While DSB is repaired mainly through homologous recombination and nonhomologous end joining in eukaryotes, DNA-PK is shown to be essential in the latter pathway. Moreover, DNA-PK is reported to be capable of phosphorylating a number of proteins, suggesting versatile functions of DNA-PK in cellular response to DSB. Here, we review the advance in our understanding on DNA-PK in three decades and remaining problems.

Keywords: DNA double-strand break repair, DNA-dependent protein kinase, Ku, nonhomologous end joining, protein phosphorylation

How to cite this article:
Matsumoto Y, Sharma MK. DNA-Dependent protein kinase in DNA damage response: Three decades and beyond. J Radiat Cancer Res 2020;11:123-34

How to cite this URL:
Matsumoto Y, Sharma MK. DNA-Dependent protein kinase in DNA damage response: Three decades and beyond. J Radiat Cancer Res [serial online] 2020 [cited 2021 Apr 17];11:123-34. Available from: https://www.journalrcr.org/text.asp?2020/11/4/123/305731

  Introduction Top

Double-strand break (DSB) is considered the most deleterious damage on DNA. It is estimated that approximately 40 DSBs are produced in a mammalian cell by each Gy of low linear energy transfer (LET) ionizing radiation (IR) such as X-ray and η-ray.[1] In addition, DSBs, as well as other types of DNA damages, are constantly generated endogenously due to reactive oxygen species, replication errors, and recombination. Cells elicit various protective measures against DNA damage, such as repair, cell cycle checkpoint, and apoptosis. To activate these measures, there are molecular sensors for each type of DNA damage. DNA-dependent protein kinase (DNA-PK) is considered a molecular sensor for DSB.

  Historical Perspectives Top

In 1985, Anderson et al. described the phosphorylation of 90 kDa heat shock protein (HSP90) in a manner dependent on double-stranded DNA (dsDNA) in the extracts of HeLa cell, rabbit reticulocyte, frog egg, and sea urchin egg.[2] This activity was purified from HeLa cell nuclei and found associated with a 300–350 kDa protein, which is now termed DNA-PK catalytic subunit (DNA-PKcs).[3],[4] Arias et al. identified a kinase activity for heptapeptide repeats in the C-terminal domain of the largest subunit of RNA polymerase II.[5] They purified this activity and showed that it consists of two components, i.e., DNA-PKcs and Ku.[6],[7] Jackson et al. found that the transcription factor Sp1 could be phosphorylated by DNA-PK in vitro in a manner dependent on Sp1 binding to GC box.[8] They studied the DNA-binding property of DNA-PK and found that it binds to and requires the end of dsDNA for activity.[9] As this property resembled Ku, they demonstrated that Ku is an essential component of DNA-PK.[9]

Ku was initially found by Mimori et al. as the antigen of autoantibody in a patient of an autoimmune disease, polymyositis-scleroderma overlap.[10] It was suggested that the antigen is a DNA-binding protein, consisting of two polypeptides, which are now termed Ku70 and Ku80 (also termed Ku86), respectively.[11] Subsequent biochemical analyses, including DNA footprinting analysis, of immunopurified Ku suggested Ku binds to the end of dsDNA, without particular preference in the nucleotide sequences.[12] In this report, they discussed a possible role of Ku in DNA repair or transposition.[12]

Initial studies also brought a considerable amount of knowledge on the properties of kinase activities. Lees-Miller and Anderson identified two threonines in the N-terminus of HSP90α as the phosphorylation sites by DNA-PK, leading to the presumption that DNA-PK preferentially phosphorylates serines or threonines followed by glutamine.[13] Their subsequent identification of DNA-PK phosphorylation sites in SV40 large T-antigen and p53 further reinforced this presumption and established SQ/TQ as the consensus sequence for DNA-PK.[14],[15] In the latter study, they synthesized a peptide substrate, mimicking the sequence around Ser15 of human p53, which has been used as in vitro DNA-PK substrate till now.[15] It must be noted, however, that the phosphorylation by DNA-PK is not limited to SQ/TQ motif.

In 1983, Bosma et al. established severe combined immunodeficiency (SCID) mutant mice, lacking B- and T-lymphocytes.[16] Subsequently, it was shown that SCID mice are defective in the V(D)J recombination of antigen receptor gene, especially at the final step.[17],[18] Fulop and Phillips,[19] Biedermann et al.,[20] and Hendrickson et al.[21] demonstrated increased IR sensitivity and reduced DSB repair ability of bone marrow cells and fibroblasts from SCID mice, suggesting a link between the repair of radiation-induced DSBs and V(D)J recombination of immunoglobulin and T-cell receptor genes. On the other hand, IR-sensitive mutants were isolated from cultured rodent cells.[22] These mutant cells were classified into 11 complementation groups, termed IR Group 1 to IR Group 11: the cells in the same complementation group were assumed to lack the same gene and the genes complementing the IR sensitivity of IR Group 1 to IR Group 11 were named XRCC1 to XRCC11, respectively, where XRCC stands for “X-ray cross-complementing.”[22] Cells classified into IR Groups 4, 5, and 7 showed similar characteristics, i.e., reduced ability in the joining of DSBs and defect in V(D)J recombination.[22] Murine SCID mutation was classified into IR Group 7. The positions of XRCC4, XRCC5, and XRCC7 were narrowed down to human chromosome 5q13–14, 2q33–35, and 8q11, respectively.[22]

In 1994, Getts and Stamato[23] and Rathmell and Chu,[24] employing gel shift assay, demonstrated the absence of DNA end-binding activity in cells of IR Group 5. Moreover, the Ku80 gene was mapped to human chromosome 2q33–35, while the Ku70 gene was mapped to 22q13. Taccioli et al.[25] and Smider et al.[26] showed that IR sensitivity and V(D)J recombination defect of IR Group 5 cells could be corrected by the introduction of Ku80 cDNA. Later, inactivating mutations were found in Ku80 gene of cells in IR Group 5 cells.[22] These studies in the aggregate proved that XRCC5 is Ku80. Ku70 is sometimes referred to as XRCC6. Unlike the case of Ku80, cell lines deficient in Ku70 have not been found in screening for IR-sensitive mutants.[22] Nevertheless, Ku70 knockout cell, which was generated by gene targeting, showed hypersensitivity to IR and V (D) J recombination defect.[22]

In 1995, Blunt et al.,[27] Kirchgessner et al.,[28] and Peterson et al.[29] demonstrated lines of evidence “strongly indicating” that XRCC7 is equivalent to DNA-PKcs. At that time, cDNA of DNA-PKcs has not been cloned yet. In addition, it could have been difficult to introduce it for expression due to its unusually large size. They demonstrated the absence of DNA-PKcs protein, its catalytic activity, and/or DNA end-binding activity in the fibroblasts from SCID mice and their presence in hybrid, in which IR resistance and V(D)J recombination were restored. DNA-PKcs gene was mapped to human chromosome 8.[30],[31] Later, nonsense mutation was identified, which removes 2% of the C-terminal region.[32] Thus, the convergence of genetic and biochemical approaches in mammalian cells greatly promoted the identification of DNA-PKcs and Ku as the critical factors in DSB repair and V(D)J recombination.

  Structure of Ku and DNA-Dependent Protein Kinase Catalytic Subunit Top

Ku70 and Ku80 consist of 609 and 732 amino acids, respectively [Figure 1]a.[33],[34] Although not noticed initially, Ku70 and Ku80 were found to have low but significant degree of similarity in primary structure, suggesting common evolutional origin.[35],[36] Indeed, in bacterial and archaebacterial organisms, Ku exists as a homodimer, not heterodimer, encoded by a single gene, not separate two genes.[37] X-ray crystallography revealed that Ku70 and Ku80 form a ring-shaped structure, encircling dsDNA, which explains dsDNA end-specific binding of Ku [Figure 1]b.[38] Ku70 and Ku80 consist of von Willebrand factor A (VWA) domain in the N-terminal region, core domain in the middle region, and C-terminal region [Figure 1a]. VWA domains mediate interaction with proteins such as XRCC4-like factor (XLF), paralog of XRCC4 and XLF (PAXX), aprataxin and polynucleotide kinase/phosphatase (PNKP)-like factor (APLF), modulator of retrovirus infection (MRI, also known as cell cycle regulator of nonhomologous end joining [NHEJ]), and Werner syndrome protein (WRN).[39] The core domains of Ku70 and Ku80 associate with each other to form the dimer and the ring.[40] The C-terminal regions of Ku70 and Ku80 are distinct from each other. The C-terminal region of Ku70 bears SAP (SAF-A/B, Acinus and PIAS) domain, which is thought to mediate DNA binding.[40] The C-terminal region of Ku80, especially 12 amino acids located at the extreme C-terminus, mediates interaction with DNA-PKcs.[36],[41] These amino acids are unique to and highly conserved in vertebrates,[41] suggesting an evolutional relationship to DNA-PKcs, which is mostly limited to vertebrates.
Figure 1: Structure of DNA-dependent protein kinase. (a) domain structure of human Ku70 and Ku80. (b) 3D-structure of Ku70 (green)-Ku80 (brown) heterodimer revealed by X-ray crystallography (PDB 1JEQ).[31] (c) domain structure of human DNA-dependent protein kinase catalytic subunit and PIKK. (d) 3D-structure of DNA-dependent protein kinase catalytic subunit revealed by X-ray crystallography (PDB 3KGV)[44]

Click here to view

Molecular cloning of cDNA for DNA-PKcs was reported in 1995 and showed that it consists of 4128 amino acids with similarity to phosphatidylinositol 3-kinase and Ataxia-telangiectasia mutated (ATM) protein in the catalytic domain located at the C-terminus [Figure 1]c.[42] Now, six members of PIKK family are known in human [Figure 1]c: DNA-PKcs, ATM,[43] ATM-and Rad3-related (ATR),[44],[45] suppressor of morphological defects on genitalia-1 (SMG-1),[46],[47] mammalian target of rapamycin (mTOR),[48],[49] and transformation/transcription domain-associated protein (TRRAP).[50] ATM and ATR are implicated in cell cycle checkpoint.[43],[44],[45] ATM acts as another sensor for DSB with Nijmegen breakage syndrome 1 (NBS1), and ATR acts as the sensor for ssDNA with ATR-interacting protein.[41] SMG-1 is involved in nonsense-mediated decay of mRNA.[46],[47] mTOR regulates cell growth and survival sensing amino acids and growth factors.[48],[49] TRRAP is involved in chromatin modification and remodeling and interestingly lacks catalytic activity.[50] In addition to kinase catalytic domain, these molecules show similarity in FAT (FRAP, ATM, and TRRAP), PRD (PIKK-regulatory domain), and FATC (FAT C-terminal) domains [Figure 1]c. They also have less conserved HEAT (huntingtin, elongation factor 3, protein phosphatase 2A, and TOR1) repeat.

The structural analysis of DNA-PK has been challenging due to its extremely large size. Sibanda et al. obtained the DNA-PKcs structure at 6.6 Å resolution by X-ray crystallography [Figure 1]d.[51] DNA-PKcs consists of a head- or crown-like substructure and a ring-like substructure, which looks like a cradle when viewed from the side.[51] Whereas the head/crown substructure corresponds to C-terminal region, including kinase domain, the ring/cradle substructure contains of HEAT repeats.[51] The latest study by Chaplin et al. revealed the holoenzyme structure at 3.5 Å resolution by cryo-electron microscopy and showed that DNA-PK holoenzyme forms a dimer, in which C-terminal region of Ku80 interacts with DNA-PKcs in the other protomer.[52] This configuration is thought to be suitable for the juxtaposition of two DNA ends and recruitment of other proteins required for DSB repair.[52]

  Components and Mechanisms for Nonhomologous End Joining Top

DSB is repaired through NHEJ and homologous recombination (HR) in vertebrates. NHEJ may sometimes incur nucleotide deletions or insertions at the junction or joining with incorrect partner, leading to chromosomal aberrations such as deletions, inversions, or translocations. HR is generally considered more accurate than NHEJ, because the sequence around the broken site is reconstituted by using homologous chromosomes and sister chromatids. However, in vertebrates, sister chromatid but not homologous chromosome is used for HR, and thus, HR is restricted to late S and G2 phases. As the majority of the cells are in G1 or G0 phases, NHEJ is of prominent importance in vertebrate cells.

In addition to Ku80 (XRCC5) and DNA-PKcs (XRCC7), XRCC4 is implicated in NHEJ. XRCC4 was identified as a human cDNA whose expression conferred normal V(D)J recombination ability and also DSB repair activity to a cell in IR Group 4.[53] Subsequently, XRCC4 was found to be associated with and essential for the function and stability of DNA ligase IV (LIG4).[54],[55],[56] Two paralogs of XRCC4 involved in NHEJ were added after 2000. In 2006, XLF (also termed Cernunnos) was found as a protein, which interacts with XRCC4, shows overall 3D structure with XRCC4, and is mutated in a patient of combined immunodeficiency.[57],[58] In 2015, three groups identified PAXX (also termed XLS for XRCC4-like small molecule).[59],[60],[61] There are two further XRCC4 paralogs, i.e., SAS6 and CCDC61 (also known as VFL3), which function in centrosome regulation.[62] The common features of XRCC4 paralogs are that they consist of the globular head at the N-terminus and flanking stalk domain and that they form homodimers through coiled-coil interaction in the latter domain.

NHEJ processes can be divided into three stages, i.e., (i) recognition, (ii) processing, and (iii) ligation of DNA ends [Figure 2]. In the recognition stage, Ku first binds to DSB and then recruits DNA-PKcs and other proteins, such as XLF, PAXX, APLF, MRI, and WRN. PAXX is shown to stabilize the binding of Ku to DSB and the NHEJ complex.[59],[60],[61] In the ligation stage, LIG4, in association with XRCC4, joins two DNA ends. XLF is thought to form filaments with XRCC4, which may have the role as align or bridge two DNA ends.[63],[64],[65],[66] The processing stage is considered essential when DNA ends are not ready for ligation. Artemis, initially identified as the causative gene for human radiosensitive-SCID,[67] forms a complex with DNA-PKcs and exerts a 5' to 3' exonuclease activity and an endonuclease activity to open a hairpin structure, which is produced at the coding end in V(D)J recombination.[68] DNA polymerase η (Polη) and DNA polymerase μ are thought to fill in the gap.[69],[70] PNKP has a dual enzymatic activity to add a phosphate group to 5' end if it is absent and to remove the phosphate group present at 3' end.[71] Aprataxin, initially identified as the causative gene for ataxia with oculomotor apraxia, removes AMP from abortive intermediates of ligation.[72] Tyrosyl-DNA phosphodiesterase 1 (TDP1) and TDP2 are thought to remove protein-DNA complex and 3'-phosphoglycolate structure.[73],[74]
Figure 2: Factors and mechanisms for nonhomologous end joining. In the recognition stage (i), Ku first binds to the double-strand break and then, recruits DNA-dependent protein kinase catalytic subunit. Paralog of XRCC4 and XRCC4-like factor stabilizes the binding of Ku to double-strand break and the assembly of other nonhomologous end joining components. The processing stage (ii) is required when DNA ends are not ready for ligation. Enzymes, such as Artemis, Polγ, Polμ, polynucleotide kinase/phosphatase, aprataxin, and TDP1, are involved depending on the structure of DNA ends. In the ligation stage (iii), LIG4 joins two DNA ends, facilitated by XRCC4 and XRCC4-like factor

Click here to view

  Role of Protein Phosphorylation by DNA-Dependent Protein Kinase in Nonhomologous End Joining Top

It was shown that catalytically inactive form of DNA-PKcs cannot restore the radioresistance and V(D)J recombination capability to DNA-PKcs-deficient cells.Äsl211Äslmult0[75],[76] This is the strongest evidence indicating that the kinase activity of DNA-PKcs essential for NHEJ. Then, which protein(s) should be phosphorylated by DNA-PK in NHEJ? And why the phosphorylation of some protein(s) by DNA-PK is essential for NHEJ? This might have looked like a simple question but is not sufficiently addressed yet. Nevertheless, there are extensive studies to identify and analyze the phosphorylation sites in core NHEJ factors, processing factors, and related factors.

The best-studied substrate of phosphorylation by DNA-PK is DNA-PKcs itself. The autophosphorylation of DNA-PKcs was found during its purification.[3],[4] The first autophosphorylation site, Thr2609, was reported by Chan et al. in 2002.[77] Subsequently, Douglas et al. identified seven autophosphorylation sites, six of which cluster around Thr2609, termed ABCDE cluster.[78],[79] Then, another clustered phosphorylation site, termed PQR cluster, was found around Ser2056.[80],[81] Now, more than 40 serines/threonines are shown to undergo autophosphorylation in vitro.[82],[83] Many of them are phosphorylated in response to IR or DNA damaging agents and essential for DNA repair function.[78],[79],[80],[81],[82],[83] However, a number of phosphorylation sites, including those in ABCDE cluster, are likely phosphorylated by ATM or ATR rather than DNA-PKcs,[84] whereas those in PQR clusters seems to be really autophosphorylated.[80],[81] The phosphorylation-specific antibody for Ser2056 has been the most commonly used as the probe for DNA-PK activity in situ. The phosphorylation in ABCDE cluster is shown to be essential for the conformational change,[85],[86] stimulation of Artemis endonuclease activity,[87] and/or the pathway choice between NHEJ and HR.[81] Because of the presence of many phosphorylation sites, further studies are required to fully understand the role of DNA-PKcs phosphorylation in NHEJ and other DNA damage responses.

Ku70 and Ku80 were also found to be substrates for DNA-PK in an initial study.[4] Ser6 of Ku70 and Ser577, Ser580, and Thr715 of Ku80 are identified as the phosphorylation sites in vitro.[88] Although these sites are phosphorylated in cellulo, the phosphorylation is constitutive and does not require DNA-PKcs (and ATM).[89] Furthermore, the mutants lacking these phosphorylation sites are functional in radioresistance and V(D)J recombination, and thus, the significance of phosphorylation is not clear.[89] Lee et al. showed the evidence suggesting that phosphorylation between Ser305 and Thr316 in Ku70 promotes the dissociation of Ku from DNA and facilitates HR.[90] However, the phosphorylation status of these phosphorylation sites and the role of DNA-PKcs therein are not clear yet.

XRCC4 was shown to be phosphorylated by DNA-PK in vitro.[55],[91],[92] Subsequently, XRCC4 was shown to be phosphorylated in cellulo in response to IR in a manner dependent on DNA-PKcs.[93] Yu et al.[94] and Lee et al.[95] identified Ser260 and Ser320 (also termed as Ser318, reflecting the alternatively spliced form) as the major phosphorylation sites in XRCC4 by purified DNA-PK in vitro. However, the XRCC4 mutants lacking these phosphorylation sites are competent in restoration of radioresistance and V(D)J recombination in XRCC4-deficient cells and also in DNA joining reaction in cell-free system, leading to the conclusion that XRCC4 phosphorylation by DNA-PK is unnecessary for these functions.[94],[95] Rather recently, it was shown that these sites are phosphorylated in response to radiation in cellulo by DNA-PK and the disruption of the latter site resulted in slight but significant increase in radiosensitivity and decrease in DSB repair ability.[96],[97] However, there are additional phosphorylation sites in XRCC4 by DNA-PK, requiring further studies to explore the significance of phosphorylation (our unpublished results). Normanno et al.[98] replaced eight potential phosphorylation sites (Ser193, Ser260, Ser304, Ser315, Ser320, Thr323, Ser327, and Ser328) in XRCC4 into alanine, to block phosphorylation, and into aspartate, to mimic phosphorylation and found that none of them could fully restore radioresistance of XRCC4-deficient cells. It was also shown that the phospho-mimic ablated the bridging of DNA in cooperation with XLF and facilitated the dissociation of XRCC4-XLF complex from DNA.[98] These phosphorylation sites, except for Ser193, are located in the intrinsically disordered C-terminal region of XRCC4. Moreover, four of them are clustered in the XRCC4 extremely C-terminal region, which is unique to and highly conserved among vertebrates.[99]

LIG4 was shown to be phosphorylated by DNA-PK at Thr650, as well as Ser668 and Ser672.[100] Although the phosphorylation at these sites is dispensable for LIG4 function, it might regulate LIG4 stability.[100] XLF was also shown to be phosphorylated by DNA-PK in vitro, and Ser245 and Ser251 were identified as the major phosphorylation sites.[101] In cellulo, Ser245 is phosphorylated by DNA-PK, whereas Ser251 is phosphorylated by ATM.[101] However, XLF mutant lacking these phosphorylation sites is competent in the restoration of radioresistance and DSB repair ability to XLF-deficient cells, indicating that phosphorylation of XLF at these sites by DNA-PK is dispensable for DNA repair function.[101] Normanno et al.[98] also mutated six putative phosphorylation sites (Ser132, Ser203, Ser245, Ser251, Ser263, and Ser266) in XLF into alanine and aspartate and showed that none of them could fully restore radioresistance of XLF-deficient cells.[98] As in the case of XRCC4, the phosphor-mimic of XLF reduced the bridging of DNA in cooperation with XRCC4 and facilitated the dissociation of XRCC4-XLF complex from DNA.[98]

Whereas Ku-deficient cells and animals are defective in both of signal joint and coding joint in V(D)J recombination, DNA-PKcs-deficient cells and animals are, at least partially, capable of signal joint.[25],[26],[27],[28] In coding joint, hairpin DNA end, generated by RAG1/2 endonuclease, needs to be opened. DNA-PKcs is not existent in many nonvertebrate organisms, suggesting that DNA-PKcs is required for a subset of NHEJ. Taken together, DNA-PKcs may play an essential role in NHEJ of “difficult” or “dirty” ends, which need processing.

Artemis is phosphorylated by DNA-PK on 11 sites in vitro.[102] In cellulo, Artemis is mainly phosphorylated by ATM rather than DNA-PK.[103],[104],[105] PNKP is also shown to be phosphorylated on Ser114 and, to a lesser extent, Ser126 by DNA-PK and ATM in vitro.[106] Whereas Ser114 is mainly phosphorylated by ATM in cellulo, DNA-PK has a partial contribution to the phosphorylation of Ser126.[106] Phospho-deficient and phospho-mimic mutants showed reduced kinase, phosphatase, and DNA binding activities.[106] This study also showed the requirement for DNA-PK and ATM in the recruitment of PNKP to DNA damage sites induced by laser.[106] The latest study using the extract of Xenopus laevis egg showed the requirement for DNA-PK catalytic activity in the processing by Polη and Tdp1.[107] Identification of the sites of phosphorylation on these proteins is awaited.

WRN is the product of the gene for Werner syndrome, which shows accelerated aging. WRN is a member of RecQ-related helicases and also has exonuclease activity. WRN interacts with Ku,[39] and DNA-PK is shown to phosphorylate WRN and to inhibit its helicase and exonuclease activities.[108],[109] Later study identified Ser440 and Ser467 as the phosphorylation sites by DNA-PK in vitro and in cellulo and showed their importance in relocalization of WRN to nucleoli.[110] Nuclear orphan receptor 4A2 (NR4A2) is phosphorylated on Ser337 by DNA-PK in vitro and in cellulo.[111] Moreover, knocking down or knocking out of NR4A2 gene and mutating Ser337 into alanine decreased DSB repair ability, indicating the importance of NR4A2 and the phosphorylation, but their precise roles need to be addressed in future studies.[111]

  Role of Protein Phosphorylation by DNA-Dependent Protein Kinase in Response to Double-Strand Break Top

Before the finding that DNA-PK and Ku are essential for DSB repair, early studies revealed that DNA-PK could phosphorylate, in vitro, a number of DNA-binding proteins, which are involved in transcription and DNA replication. Therefore, possible roles of DNA-PK in the regulation of transcription and DNA replication were considered.[112] There are also a number of proteins with diverse cellular functions, which were shown to be phosphorylated by DNA-PK in vitro and in cellulo [Figure 3]. Below, we will introduce some of them and the role of DNA-PK in response to DSB, not limited to NHEJ.
Figure 3: In vitro and in cellulo substrates for DNA-dependent protein kinase. DNA-dependent protein kinase is shown to phosphorylate a number of proteins involved in DNA transactions, such as DNA repair, DNA replication, transcription, and chromatin regulation, and also proteins with other diverse functions, such as the regulation of organelle and cytoskeleton. Proteins described in the text are colored green and those not described are colored orange. Proteins which have not yet been reported to be phosphorylated by DNA-dependent protein kinase are colored gray

Click here to view

p53, the gene of which is frequently mutated in cancer, is upregulated in response to DNA damage and regulates the expression of genes involved in cell cycle checkpoint and apoptosis. Initial studies showed that p53 is phosphorylated by DNA-PK on Ser15 and Ser37.[4],[16] The phosphorylation of p53 on Ser15 and Ser37 reduces its interaction with murine double minute 2, which inhibits and promotes degradation of p53 through ubiquitylation.[113] However, it was subsequently shown that ATM rather than DNA-PK is mainly responsible for DSB-induced phosphorylation of p53 on Ser15.[114],[115] DNA-PK also phosphorylates Ser9 and Ser46 of p53, but ATM rather than DNA-PK is mainly responsible for IR-induced phosphorylation in cellulo.[116],[117]

Replication protein A (RPA) consists of three subunits of 70 kDa, 30 kDa, and 14 kDa, which are called RPA1, RPA2, and RPA3, respectively. RPA binds to single-strand DNA and exerts multiple functions in DNA replication, recombination, repair, and cell cycle checkpoint. DNA-PK phosphorylates Ser4, Ser8, Ser12, Thr21, and Ser33 in vitro.[118],[119] Not only DNA-PK but also ATM and ATR are thought to phosphorylate these sites in cellulo. DNA-PK is essential for the phosphorylation of Ser4 and/or Ser8 in replication stress and suppresses inaccurate HR and mitotic catastrophe through regulation of fork restart and origin firing.[120]

Histone H2AX is one of the variants of histone H2A, representing 2%–25% of total H2A. Ser139 of H2AX undergoes phosphorylation in response to DSB, which is called η-H2AX.[121] The phosphorylation is induced in wide region of megabase size around each DSB.[122] Now, η-H2AX is the most frequently used indicator for DSB and its repair. H2AX Ser139 is phosphorylated by DNA-PK as well as ATM.[123],[124] η-H2AX binds to mediator of DNA damage checkpoint protein 1, which in turn recruits a series of proteins involved in DSB repair and cell cycle checkpoint through direct or indirect protein-protein interactions.[125]

Valosin-containing protein (VCP, also known as p97) is shown to be phosphorylated on Ser784 by DNA-PK in vitro and in cellulo, although in cellulo phosphorylation is mediated also by ATM and ATR, depending on the timing and type of DNA damage.[126] VCP is a member of AAA+ (ATPases associated with diverse cellular activities) ATPase and a protein chaperon regulating folding and translocation of proteins. Ser784-phosphorylated VCP accumulates at DSB. VCP is shown to extract sterically trapped Ku ring[127] and also promote degradation of DNA-PKcs.[128] Although VCP seems to be deeply implicated in NHEJ, the role of phosphorylation on Ser784 in NHEJ remains to be clarified.

DNA-PK is shown to phosphorylate nuclear fumarase on Thr236 in vitro and in cellulo.[129] This facilitates interaction of fumarase with histone H2A variant H2AZ at DSB, leading to local generation of fumarate, which inhibit histone demethylase KDM2B.[129] As a result, dimethylation of histone H3 Lys36 is enhanced and the accumulation of DNA-PKcs-Ku at DSB is increased, showing a possible feedback mechanism for DNA-PK activation at DSB.[129]

Farber-Katz et al. found that DNA damage induces dispersal of Golgi's apparatus throughout the cytoplasm.[130] Golgi phosphoprotein 3 (GOLPH 3) regulates the morphology of Golgi in cooperation with myosin 18A (MYO18A) and F-actin.[130] DNA-PK phosphorylates Thr148 of GOLPH 3, which is essential for Golgi dispersal.[130] GOLPH 3 and MYO18A are essential for cell survival after DNA damage. The biological significance of DNA damage-induced Golgi dispersal remains to be clarified.

Phosphoproteomic analyses reveal unexpected substrates of protein kinases. Matsuoka et al.[131] searched for ATM/ATR substrates in cellulo through stable amino acid labeling in cell culture followed by mass spectrometry. This study identified more than 900 phosphorylation sites on more than 700 proteins, revealing extensive network of DNA damage response. Kotula et al.[132] reported a proteomic search for DNA-PK substrates through two-dimensional electrophoresis followed by ProQ Diamond staining, leading to the identification of 26 proteins, including HSPs and cytoskeleton proteins. One of them is vimentin, which is phosphorylated on Ser459. It is suggested that DNA damage-induced phosphorylation of vimentin suppresses cell motility and adhesion.[132] Thus, DNA-PK might regulate a broad range of response to DSB through phosphorylation.

  Concluding Remarks and Future Directions Top

Here, we have reviewed the history of research on DNA-PK in three decades. We have got a clear view that DNA-PK, composed of DNA-PKcs and Ku70/Ku80, plays a pivotal role in DSB repair through NHEJ, in cooperation with XRCC4, LIG4, XLF, PAXX, and processing enzymes. However, the role of the phosphorylation in NHEJ has remained as a great missing link. Although a number of phosphorylation sites in core NHEJ factors and associated factors have been identified and analyzed, further studies are necessary to fully understand the function of DNA-PK in NHEJ. From the evolutional aspect and the phenotype of deficient cells, it might be presumed that DNA-PKcs is especially required for the repair of “difficult” or “dirty” ends, which need processing. In this regard, possible difference in the response of DNA-PK to various types of DNA damages, e.g., low LET radiation, high LET radiation, and chemicals, can be of interest. In addition, a number of proteins, which are not currently implicated in NHEJ, are also shown to be phosphorylated in cellulo, suggesting versatile functions of DNA-PK in cellular response to DSB. Proteomic search for DNA-PK substrates in cellulo would be important to clarify the entire function of DNA-PK in response to DSB.

Although here we have focused mainly on DNA-PK function, DNA-PK has great implications in carcinogenesis and cancer radiotherapy. There are a number of animal and human studies showing that the loss or reduction in DNA-PK or Ku is associated with increase in genomic instability and cancer risk. On the other hand, there are also studies showing upregulation of DNA-PKcs or Ku in various types of human cancer. In addition, a number of studies have shown the correlation between the expression of DNA-PKcs or Ku and cancer prognosis or therapeutic responses, underscoring the usefulness of DNA-PK or Ku as predicative biomarkers. Finally, DNA-PK is a promising target for sensitization of cancer cells to radiotherapy and anticancer agent treatment. These aspects are reviewed elsewhere.[133] Thus, the understanding of the molecular property and function of DNA-PK will provide approaches to the prediction of cancer risk and prognosis and also to the enhancement of cancer treatment.

Financial support and sponsorship

This study was financially supported by Grant-in-Aid for Scientific Research (24390290, 15H02817, 20H04334) and Challenging Research (25550024, 17K20042) from Japan Society for the Promotion of Science (JSPS) to YM, Takeda Science Foundation, JSPS, and Tokyo Biochemical Research Foundation to MKS.

Conflicts of interest

There are no conflicts of interest.

  References Top

United Nations Scientific Committee on the Effects of Atomic Radiation. UNSCEAR 2000 Report to the General Assembly, with Scientific Annexes Volume II: Effects 4; 2000.  Back to cited text no. 1
Walker AI, Hunt T, Jackson RJ, Anderson CW. Double-stranded DNA induces the phosphorylation of several proteins including the 90 000 mol. Wt. Heat-shock protein in animal cell extracts. EMBO J 1985;4:139-45.  Back to cited text no. 2
Carter T, Vancurová I, Sun I, Lou W, DeLeon S. A DNA-activated protein kinase from heLa cell nuclei. Mol Cell Biol 1990;10:6460-71.  Back to cited text no. 3
Lees-Miller SP, Chen YR, Anderson CW. Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human ku autoantigen. Mol Cell Biol 1990;10:6472-81.  Back to cited text no. 4
Arias JA, Peterson SR, Dynan WS. Promoter-dependent phosphorylation of RNA polymerase II by a template-bound kinase. Association with transcriptional initiation. J Biol Chem 1991;266:8055-61.  Back to cited text no. 5
Dvir A, Peterson SR, Knuth MW, Lu H, Dynan WS. Ku autoantigen is the regulatory component of a template-associated protein kinase that phosphorylates RNA polymerase II. Proc Natl Acad Sci U S A 1992;89:11920-4.  Back to cited text no. 6
Dvir A, Stein LY, Calore BL, Dynan WS. Purification and characterization of a template-associated protein kinase that phosphorylates RNA polymerase II. J Biol Chem 1993;268:10440-7.  Back to cited text no. 7
Jackson SP, MacDonald JJ, Lees-Miller S, Tjian R. GC box binding induces phosphorylation of sp1 by a DNA-dependent protein kinase. Cell 1990;63:155-65.  Back to cited text no. 8
Gottlieb TM, Jackson SP. The DNA-dependent protein kinase: Requirement for DNA ends and association with ku antigen. Cell 1993;72:131-42.  Back to cited text no. 9
Mimori T, Akizuki M, Yamagata H, Inada S, Yoshida S, Homma M, et al. Characterization of a high molecular weight acidic nuclear protein recognized by autoantibodies in sera from patients with polymyositis-scleroderma overlap. J Clin Invest 1981;68:611-20.  Back to cited text no. 10
Mimori T, Hardin JA, Steitz JA. Characterization of the DNA-binding protein antigen ku recognized by autoantibodies from patients with rheumatic disorders. J Biol Chem 1986;261:2274-8.  Back to cited text no. 11
Mimori T, Hardin JA. Mechanism of interaction between Ku protein and DNA. J Biol Chem 1986;261:10375-9.  Back to cited text no. 12
Lees-Miller SP, Anderson CW. Two human 90-kDa heat shock proteins are phosphorylated in vivo at conserved serines that are phosphorylated in vitro by casein kinase II. J Biol Chem 1989;264:2431-7.  Back to cited text no. 13
Chen YR, Lees-Miller SP, Tegtmeyer P, Anderson CW. The human DNA-activated protein kinase phosphorylates simian virus 40 T antigen at amino- and carboxy-terminal sites. J Virol 1991;65:5131-4.  Back to cited text no. 14
Lees-Miller SP, Sakaguchi K, Ullrich SJ, Appella E, Anderson CW. Human DNA-activated protein kinase phosphorylates serines 15 and 37 in the amino-terminal transactivation domain of human p53. Mol Cell Biol 1992;12:5041-9.  Back to cited text no. 15
Bosma GC, Custer RP, Bosma MJ. A severe combined immunodeficiency mutation in the mouse. Nature 1983;301:527-30.  Back to cited text no. 16
Schuler W, Weiler IJ, Schuler A, Phillips RA, Rosenberg N, Mak TW, et al. Rearrangement of antigen receptor genes is defective in mice with severe combined immune deficiency. Cell 1986;46:963-72.  Back to cited text no. 17
Malynn BA, Blackwell TK, Fulop GM, Rathbun GA, Furley AJ, Ferrier P, et al. The scid defect affects the final step of the immunoglobulin VDJ recombinase mechanism. Cell 1988;54:453-60.  Back to cited text no. 18
Fulop GM, Phillips RA. The scid mutation in mice causes general defect in DNA repair. Nature 1990;347:479-82.  Back to cited text no. 19
Biedermann KA, Sun J, Giaccia AJ, Tosto LM, Brown JM. Scid mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double-strand break repair. Proc Natl Acad Sci U S A 1991;88:1394-7.  Back to cited text no. 20
Hendrickson EA, Qin XQ, Bump EA, Schatz DG, Oettinger M, Weaver DT. A link between double-strand break-related repair and V (D) J recombination: The scid mutation. Proc Natl Acad Sci U S A 1991;88:4061-5.  Back to cited text no. 21
Thacker J, Zdzienicka MZ. The mammalian XRCC genes: their roles in DNA repair and genetic integrity. DNA Repair 2003;2:655-72.  Back to cited text no. 22
Getts RC, Stamato TD. Absence of a ku-like DNA end binding activity in the xrs double-strand DNA repair-deficient mutant. J Biol Chem 1994;269:15981-4.  Back to cited text no. 23
Rathmell WK, Chu G. Involvement of the ku autoantigen in the cellular response to DNA double-strand breaks. Proc Natl Acad Sci U S A 1994;91:7623-7.  Back to cited text no. 24
Taccioli GE, Gottlieb TM, Blunt T, Priestley A, Demengeot J, Mizuta R, et al. Ku80: Product of the XRCC5 gene and its role in DNA repair and V (D) J recombination. Science 1994;265:1442-5.  Back to cited text no. 25
Smider V, Rathmell WK, Lieber MR, Chu G. Restoration of X-ray resistance and V (D) J recombination in mutant cells by ku cDNA. Science 1994;266:288-91.  Back to cited text no. 26
Blunt T, Finnie NJ, Taccioli GE, Smith GC, Demengeot J, Gottlieb TM, et al. Defective DNA-dependent protein kinase activity is linked to V (D) J recombination and DNA repair defects associated with the murine scid mutation. Cell 1995;80:813-23.  Back to cited text no. 27
Kirchgessner CU, Patil CK, Evans JW, Cuomo CA, Fried LM, Carter T, et al. DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect. Science 1995;267:1178-83.  Back to cited text no. 28
Peterson SR, Kurimasa A, Oshimura M, Dynan WS, Bradbury EM, Chen DJ, et al. Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells. Proc Natl Acad Sci U S A 1995;92:3171-4.  Back to cited text no. 29
Sipley JD, Menninger JC, Hartley KO, Ward DC, Jackson SP, Anderson CW, et al. Gene for the catalytic subunit of the human DNA-activated protein kinase maps to the site of the XRCC7 gene on chromosome 8. Proc Natl Acad Sci U S A 1995;92:7515-9.  Back to cited text no. 30
Miller RD, Hogg J, Ozaki JH, Gell D, Jackson SP, Riblet R, et al. Gene for the catalytic subunit of mouse DNA-dependent protein kinase maps to the scid locus. Proc Natl Acad Sci U S A 1995;92:10792-5.  Back to cited text no. 31
Blunt T, Gell D, Fox M, Taccioli GE, Lehmann AR, Jackson SP, et al. Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse. Proc Natl Acad Sci U S A 1996;93:10285-90.  Back to cited text no. 32
Reeves WH, Sthoeger ZM. Molecular cloning of cDNA encoding the p70 (Ku) lupus autoantigen. J Biol Chem 1989;264:5047-52.  Back to cited text no. 33
Yaneva M, Wen J, Ayala A, Cook R. CDNA-derived amino acid sequence of the 86-kDa subunit of the ku antigen. J Biol Chem 1989;264:13407-11.  Back to cited text no. 34
Dynan WS, Yoo S. Interaction of ku protein and DNA-dependent protein kinase catalytic subunit with nucleic acids. Nucleic Acids Res 1998;26:1551-9.  Back to cited text no. 35
Gell D, Jackson SP. Mapping of protein-protein interactions within the DNA-dependent protein kinase complex. Nucleic Acids Res 1999;27:3494-502.  Back to cited text no. 36
Aravind L, Koonin EV. Prokaryotic homologs of the eukaryotic DNA-end-binding protein ku, novel domains in the ku protein and prediction of a prokaryotic double-strand break repair system. Genome Res 2001;11:1365-74.  Back to cited text no. 37
Walker JR, Corpina RA, Goldberg J. Structure of the Ku heterodimer bound to DNA and its implication for double-strand break repair. Nature 2001;412:607-14.  Back to cited text no. 38
Kim K, Min J, Kirby TW, Gabel SA, Pedersen LC, London RE, et al. Ligand binding characteristics of the ku80 von willebrand domain. DNA Repair (Amst) 2020;85:102739.  Back to cited text no. 39
Inagawa T, Wennink T, Lebbink JHG, Keijzers G, Florea BI, Verkaik NS, et al. C-terminal extensions of ku70 and ku80 differentially influence DNA end binding properties. Int. J. Mol. Sci. 2020;21:6725.  Back to cited text no. 40
Falck J, Coates J, Jackson SP. Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage. Nature 2005;434:605-11.  Back to cited text no. 41
Hartley KO, Gell D, Smith GC, Zhang H, Divecha N, Connelly MA, et al. DNA-dependent protein kinase catalytic subunit: A relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product. Cell 1995;82:849-56.  Back to cited text no. 42
Savitsky K, Bar-Shira A, Gilad S, Rotman G, Ziv Y, Vanagaite L, et al. A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science 1995;268:1749-53.  Back to cited text no. 43
Bentley NJ, Holtzman DA, Flaggs G, Keegan KS, DeMaggio A, Ford JC, et al. The schizosaccharomyces pombe rad3 checkpoint gene. EMBO J 1996;15:6641-51.  Back to cited text no. 44
Cimprich KA, Shin TB, Keith CT, Schleiber SL. cDNA cloning and gene mapping of a candidate human cell cycle checkpoint protein. Proc Natl Acad Sci U S A 1996;93:2850-5.  Back to cited text no. 45
Denning G, Jamieson L, Maquat LE, Thompson EA, Fields AP. Cloning of a novel phosphatidylinositol kinase-related kinase: characterization of the human SMG-1 RNA surveillance protein. J Biol Chem 2001;276:22709-14.  Back to cited text no. 46
Yamashita A, Ohnishi T, Kashima I, Taya Y, Ohno S. Human SMG-1, a novel phosphatidylinositol 3-kinase-related protein kinase, associates with components of the mRNA surveillance complex and is involved in the regulation of nonsense-mediated mRNA decay. Genes Dev 2001;15:2215-28.  Back to cited text no. 47
Brown EJ, Albers MW, Shin TB, Ichikawa K, Keith CT, Lane WS, et al. A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature 1994;369:756-8.  Back to cited text no. 48
Sabatini DM, Erdjument-Bromage H, Lui M, Tempst P, Snyder SH. RAFT1: A mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell 1994;78:35-43.  Back to cited text no. 49
McMahon SB, Van Buskirk HA, Dugan KA, Copeland TD, Cole MD. The novel ATM-related protein TRRAP is an essential cofactor for the c-myc and E2F oncoproteins. Cell 1998;94:363-74.  Back to cited text no. 50
Sibanda BL, Chirgadze DY, Blundell TL. Crystal structure of DNA-PKcs reveals a large open-ring cradle comprised of HEAT repeats. Nature 2010;463:118-21.  Back to cited text no. 51
Chaplin AK, Hardwick SW, Liang S, Kefala Stavridi A, Hnizda A, Cooper LR, et al. Dimers of DNA-PK create a stage for DNA double-strand break repair. Nat Struct Mol Biol 2020. doi: 10.1038/s41594-020-00517-x.  Back to cited text no. 52
Li Z, Otevrel T, Gao Y, Cheng HL, Seed B, Stamato TD, et al. The XRCC4 gene encodes a novel protein involved in DNA double-strand break repair and V (D) J recombination. Cell 1995;83:1079-89.  Back to cited text no. 53
Grawunder U, Wilm M, Wu X, Kulesza P, Wilson TE, Mann M, et al. Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells. Nature 1997;388:492-5.  Back to cited text no. 54
Critchlow SE, Bowater RP, Jackson SP. Mammalian DNA double-strand break repair protein XRCC4 interacts with DNA ligase IV. Curr Biol 1997;7:588-98.  Back to cited text no. 55
Bryans M, Valenzano MC, Stamato TD. Absence of DNA ligase IV protein in XR-1 cells: Evidence for stabilization by XRCC4. Mutat Res 1999;433:53-8.  Back to cited text no. 56
Ahnesorg P, Smith P, Jackson SP. XLF interacts with the XRCC4-DNA ligase IV complex to promote DNA nonhomologous end-joining. Cell 2006;124:301-13.  Back to cited text no. 57
Buck D, Malivert L, de Chasseval R, Barraud A, Fondanèche MC, Sanal O, et al. Cernunnos, a novel nonhomologous end-joining factor, is mutated in human immunodeficiency with microcephaly. Cell 2006;124:287-99.  Back to cited text no. 58
Ochi T, Blackford AN, Coates J, Jhujh S, Mehmood S, Tamura N, et al. DNA repair. PAXX, a paralog of XRCC4 and XLF, interacts with ku to promote DNA double-strand break repair. Science 2015;347:185-8.  Back to cited text no. 59
Xing M, Yang M, Huo W, Feng F, Wei L, Jiang W, et al. Interactome analysis identifies a new paralogue of XRCC4 in non-homologous end joining DNA repair pathway. Nat Commun 2015;6:6233.  Back to cited text no. 60
Craxton A, Somers J, Munnur D, Jukes-Jones R, Cain K, Malewicz M, et al. XLS (c9orf142) is a new component of mammalian DNA double-stranded break repair. Cell Death Differ 2015;22:890-7.  Back to cited text no. 61
Ochi T, Quarantotti V, Lin H, Jullien J, Rosa E Silva I, Boselli F, et al. CCDC61/VFL3 is a paralog of SAS6 and promotes ciliary functions. Structure 2020;28:674-8900000000000.  Back to cited text no. 62
Hammel M, Yu Y, Fang S, Lees-Miller SP, Tainer JA. XLF regulates filament architecture of the XRCC4·ligase IV complex. Structure 2010;18:1431-42.  Back to cited text no. 63
Ropars V, Drevet P, Legrand P, Baconnais S, Amram J, Faure G, et al. Structural characterization of filaments formed by human xrcc4-cernunnos/XLF complex involved in nonhomologous DNA end-joining. Proc Natl Acad Sci U S A 2011;108:12663-8.  Back to cited text no. 64
Andres SN, Vergnes A, Ristic D, Wyman C, Modesti M, Junop M, et al. A human XRCC4-XLF complex bridges DNA. Nucleic Acids Res 2012;40:1868-78.  Back to cited text no. 65
Mahaney BL, Hammel M, Meek K, Tainer JA, Lees-Miller SP. XRCC4 and XLF form long helical protein filaments suitable for DNA end protection and alignment to facilitate DNA double strand break repair. Biochem Cell Biol 2013;91:31-41.  Back to cited text no. 66
Moshous D, Callebaut I, de Chasseval R, Corneo B, Cavazzana-Calvo M, le Deist F, et al. Artemis, a novel DNA double-strand break repair/V (D) J recombination protein, is mutated in human severe combined immune deficiency. Cell 2001;105:177-86.  Back to cited text no. 67
Ma Y, Pannicke U, Schwarz K, Lieber MR. Hairpin opening and overhang processing by an artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V (D) J recombination. Cell 2002;108:781-94.  Back to cited text no. 68
Nick McElhinny SA, Havener JM, Garcia-Diaz M, Juárez R, Bebenek K, Kee BL, et al. A gradient of template dependence defines distinct biological roles for family X polymerases in nonhomologous end joining. Mol Cell 2005;19:357-66.  Back to cited text no. 69
Capp JP, Boudsocq F, Bertrand P, Laroche-Clary A, Pourquier P, Lopez BS, et al. The DNA polymerase lambda is required for the repair of non-compatible DNA double strand breaks by NHEJ in mammalian cells. Nucleic Acids Res 2006;34:2998-3007.  Back to cited text no. 70
Chappell C, Hanakahi LA, Karimi-Busheri F, Weinfeld M, West SC. Involvement of human polynucleotide kinase in double-strand break repair by non-homologous end joining. EMBO J 2002;21:2827-32.  Back to cited text no. 71
Ahel I, Rass U, El-Khamisy SF, Katyal S, Clements PM, McKinnon PJ, et al. The neurodegenerative disease protein aprataxin resolves abortive DNA ligation intermediates. Nature 2006;443:713-6.  Back to cited text no. 72
Nitiss KC, Malik M, He X, White SW, Nitiss JL. Tyrosyl-DNA phosphodiesterase (Tdp1) participates in the repair of top2-mediated DNA damage. Proc Natl Acad Sci U S A 2006;103:8953-8.  Back to cited text no. 73
Gómez-Herreros F, Romero-Granados R, Zeng Z, Alvarez-Quilón A, Quintero C, Ju L, et al. TDP2-dependent non-homologous end-joining protects against topoisomerase II-induced DNA breaks and genome instability in cells and in vivo. PLoS Genet 2013;9:e1003226.  Back to cited text no. 74
Kienker LJ, Shin EK, Meek K. Both V (D) J recombination and radioresistance require DNA-PK kinase activity, though minimal levels suffice for V (D) J recombination. Nucleic Acids Res 2000;28:2752-61.  Back to cited text no. 75
Kurimasa A, Kumano S, Boubnov NV, Story MD, Tung CS, Peterson SR, et al. Requirement for the kinase activity of human DNA-dependent protein kinase catalytic subunit in DNA strand break rejoining. Mol Cell Biol 1999;19:3877-84.  Back to cited text no. 76
Chan DW, Chen BP, Prithivirajsingh S, Kurimasa A, Story MD, Qin J, et al. Autophosphorylation of the DNA-dependent protein kinase catalytic subunit is required for rejoining of DNA double-strand breaks. Genes Dev 2002;16:2333-8.  Back to cited text no. 77
Douglas P, Sapkota GP, Morrice N, Yu Y, Goodarzi AA, Merkle D, et al. Identification of in vitro and in vivo phosphorylation sites in the catalytic subunit of the DNA-dependent protein kinase. Biochem J 2002;368:243-51.  Back to cited text no. 78
Ding Q, Reddy YV, Wang W, Woods T, Douglas P, Ramsden DA, et al. Autophosphorylation of the catalytic subunit of the DNA-dependent protein kinase is required for efficient end processing during DNA double-strand break repair. Mol Cell Biol 2003;23:5836-48.  Back to cited text no. 79
Chen BP, Chan DW, Kobayashi J, Burma S, Asaithamby A, Morotomi-Yano K. Cell cycle dependence of DNA-dependent protein kinase phosphorylation in response to DNA double strand breaks. J Biol Chem 2005;280:14709-15.  Back to cited text no. 80
Cui X, Yu Y, Gupta S, Cho YM, Lees-Miller SP, Meek K, et al. Autophosphorylation of DNA-dependent protein kinase regulates DNA end processing and may also alter double-strand break repair pathway choice. Mol Cell Biol 2005;25:10842-52.  Back to cited text no. 81
Neal JA, Dang V, Douglas P, Wold MS, Lees-Miller SP, Meek K, et al. Inhibition of homologous recombination by DNA-dependent protein kinase requires kinase activity, is titratable, and is modulated by autophosphorylation. Mol Cell Biol 2011;31:1719-33.  Back to cited text no. 82
Neal JA, Sugiman-Marangos S, VanderVere-Carozza P, Wagner M, Turchi J, Lees-Miller SP, et al. Unraveling the complexities of DNA-dependent protein kinase autophosphorylation. Mol Cell Biol 2014;34:2162-75.  Back to cited text no. 83
Chen BP, Uematsu N, Kobayashi J, Lerenthal Y, Krempler A, Yajima H, et al. Ataxia telangiectasia mutated (ATM) is essential for DNA-PKcs phosphorylations at the thr-2609 cluster upon DNA double strand break. J Biol Chem 2007;282:6582-7.  Back to cited text no. 84
Block WD, Yu Y, Merkle D, Gifford JL, Ding Q, Meek K, et al. Autophosphorylation-dependent remodeling of the DNA-dependent protein kinase catalytic subunit regulates ligation of DNA ends. Nucleic Acids Res 2004;32:4351-7.  Back to cited text no. 85
Reddy YV, Ding Q, Lees-Miller SP, Meek K, Ramsden DA. Non-homologous end joining requires that the DNA-PK complex undergo an autophosphorylation-dependent rearrangement at DNA ends. J Biol Chem 2004;279:39408-13.  Back to cited text no. 86
Goodarzi AA, Yu Y, Riballo E, Douglas P, Walker SA, Ye R, et al. DNA-PK autophosphorylation facilitates artemis endonuclease activity. EMBO J 2006;25:3880-9.  Back to cited text no. 87
Chan DW, Ye R, Veillette CJ, Lees-Miller SP. DNA-dependent protein kinase phosphorylation sites in ku 70/80 heterodimer. Biochemistry 1999;38:1819-28.  Back to cited text no. 88
Douglas P, Gupta S, Morrice N, Meek K, Lees-Miller SP. DNA-PK-dependent phosphorylation of Ku70/80 is not required for non-homologous end joining. DNA Repair 2005;4:1006-18.  Back to cited text no. 89
Lee KJ, Saha J, Sun J, Fattah KR, Wang SC, Jakob B, et al. Phosphorylation of ku dictates DNA double-strand break (DSB) repair pathway choice in S phase. Nucleic Acids Res 2016;44:1732-45.  Back to cited text no. 90
Leber R, Wise TW, Mizuta R, Meek K. The XRCC4 gene product is a target for and interacts with the DNA-dependent protein kinase. J Biol Chem 1998;273:1794-801.  Back to cited text no. 91
Modesti M, Hesse J, Gellert M. DNA binding of Xrcc4 protein is associated with V (D) J recombination but not with stimulation of DNA ligase IV activity. EMBO J 1999;18:2008-18.  Back to cited text no. 92
Matsumoto Y, Suzuki N, Namba N, Umeda N, Ma XJ, Morita A, et al. Cleavage and phosphorylation of XRCC4 protein induced by X-irradiation. FEBS Lett 2000;478:67-71.  Back to cited text no. 93
Yu Y, Wang W, Ding Q, Ye R, Chen D, Merkle D, et al. DNA-PK phosphorylation sites in XRCC4 are not required for survival after radiation or for V (D) J recombination. DNA Repair 2003;2:1239-52.  Back to cited text no. 94
Lee KJ, Jovanovic M, Udayakumar D, Bladen CL, Dynan WS. Identification of DNA-PKcs phosphorylation sites in XRCC4 and effects of mutation at these sites on DNA end joining in a cell-free system. DNA Repair 2003;3:267-76.  Back to cited text no. 95
Sharma MK, Imamichi S, Fukuchi M, Samarth RM, Tomita M, Matsumoto Y, et al. In cellulo phosphorylation of XRCC4 ser320 by DNA-PK induced by DNA damage. J Radiat Res 2016;57:115-20.  Back to cited text no. 96
Amiri Moghani AR, Sharma MK, Matsumoto Y. In cellulo phosphorylation of DNA double-strand break repair protein XRCC4 on ser260 by DNA-PK. J Radiat Res 2018;59:700-8.  Back to cited text no. 97
Normanno D, Négrel A, de Melo AJ, Betzi S, Meek K, Modesti M, et al. Mutational phospho-mimicry reveals a regulatory role for the XRCC4 and XLF C-terminal tails in modulating DNA bridging during classical non-homologous end joining. Elife 2017;6:e22900.  Back to cited text no. 98
Wanotayan R, Fukuchi M, Imamichi S, Sharma MK, Matsumoto Y. Asparagine 326 in the extremely C-terminal region of XRCC4 is essential for the cell survival after irradiation. Biochem Biophys Res Commun 2015;457:526-31.  Back to cited text no. 99
Wang YG, Nnakwe C, Lane WS, Modesti M, Frank KM. Phosphorylation and regulation of DNA ligase IV stability by DNA-dependent protein kinase. J Biol Chem 2004;279:37282-90.  Back to cited text no. 100
Yu Y, Mahaney BL, Yano K, Ye R, Fang S, Douglas P. DNA-PK and ATM phosphorylation sites in XLF/Cernunnos are not required for repair of DNA double strand breaks. DNA Repair 2008;7:1680-92.  Back to cited text no. 101
Ma Y, Pannicke U, Lu H, Niewolik D, Schwarz K, Lieber MR, et al. The DNA-dependent protein kinase catalytic subunit phosphorylation sites in human artemis. J Biol Chem 2005;280:33839-46.  Back to cited text no. 102
Zhang X, Succi J, Feng Z, Prithivirajsingh S, Story MD, Legerski RJ, et al. Artemis is a phosphorylation target of ATM and ATR and is involved in the G2/M DNA damage checkpoint response. Mol Cell Biol 2004;24:9207-20.  Back to cited text no. 103
Poinsignon C, de Chasseval R, Soubeyrand S, Moshous D, Fischer A, Haché RJ, et al. Phosphorylation of Artemis following irradiation-induced DNA damage. Eur J Immunol 2004;34:3146-55.  Back to cited text no. 104
Chen L, Morio T, Minegishi Y, Nakada S, Nagasawa M, Komatsu K, et al. Ataxia-telangiectasia-mutated dependent phosphorylation of artemis in response to DNA damage. Cancer Sci 2005;96:134-41.  Back to cited text no. 105
Zolner AE, Abdou I, Ye R, Mani RS, Fanta M, Yu Y, et al. Phosphorylation of polynucleotide kinase/phosphatase by DNA-dependent protein kinase and ataxia-telangiectasia mutated regulates its association with sites of DNA damage. Nucleic Acids Res 2011;39:9224-37.  Back to cited text no. 106
Stinson BM, Moreno AT, Walter JC, Loparo JJ. A mechanism to minimize errors during non-homologous end joining. Mol Cell 2020;77:1080-9100000000.  Back to cited text no. 107
Yannone SM, Roy S, Chan DW, Murphy MB, Huang S, Campisi J, et al. Werner syndrome protein is regulated and phosphorylated by DNA-dependent protein kinase. J Biol Chem 2001;276:38242-8.  Back to cited text no. 108
Karmakar P, Piotrowski J, Brosh RM Jr. Sommers JA, Miller SP, Cheng WH, et al. Werner protein is a target of DNA-dependent protein kinase in vivo and in vitro, and its catalytic activities are regulated by phosphorylation. J Biol Chem 2002;277:18291-302.  Back to cited text no. 109
Kusumoto-Matsuo R, Ghosh D, Karmakar P, May A, Ramsden D, Bohr VA, et al. Serines 440 and 467 in the werner syndrome protein are phosphorylated by DNA-PK and affects its dynamics in response to DNA double strand breaks. Aging (Albany NY) 2014;6:70-81.  Back to cited text no. 110
Malewicz M, Kadkhodaei B, Kee N, Volakakis N, Hellman U, Viktorsson K, et al. Essential role for DNA-PK-mediated phosphorylation of NR4A nuclear orphan receptors in DNA double-strand break repair. Genes Dev 2011;25:2031-40.  Back to cited text no. 111
Anderson CW, Lees-Miller SP. The nuclear serine/threonine protein kinase DNA-PK. Crit Rev Eukaryot Gene Expr 1992;2:283-314.  Back to cited text no. 112
Shieh SY, Ikeda M, Taya Y, Prives C. DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell 1997;91:325-34.  Back to cited text no. 113
Canman CE, Lim DS, Cimprich KA, Taya Y, Tamai K, Sakaguchi K, et al. Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 1998;281:1677-9.  Back to cited text no. 114
Banin S, Moyal L, Shieh S, Taya Y, Anderson CW, Chessa L, et al. Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 1998;281:1674-7.  Back to cited text no. 115
Saito S, Yamaguchi H, Higashimoto Y, Chao C, Xu Y, Fornace AJ Jr, et al. Phosphorylation site interdependence of human p53 post-translational modifications in response to stress. J Biol Chem 2003;278:37536-44.  Back to cited text no. 116
Komiyama S, Taniguchi S, Matsumoto Y, Tsunoda E, Ohto T, Suzuki Y, et al. Potentiality of DNA-dependent protein kinase to phosphorylate ser46 of human p53. Biochem Biophys Res Commun 2004;323:816-22.  Back to cited text no. 117
Niu H, Erdjument-Bromage H, Pan ZQ, Lee SH, Tempst P, Hurwitz J, et al. Mapping of amino acid residues in the p34 subunit of human single-stranded DNA-binding protein phosphorylated by DNA-dependent protein kinase and cdc2 kinase in vitro. J Biol Chem 1997;272:12634-41.  Back to cited text no. 118
Zernik-Kobak M, Vasunia K, Connelly M, Anderson CW, Dixon K. Sites of UV-induced phosphorylation of the p34 subunit of replication protein A from heLa cells. J Biol Chem 1997;272:23896-904.  Back to cited text no. 119
Ashley AK, Shrivastav M, Nie J, Amerin C, Troksa K, Glanzer JG, et al. DNA-PK phosphorylation of RPA32 ser4/Ser8 regulates replication stress checkpoint activation, fork restart, homologous recombination and mitotic catastrophe. DNA Repair (Amst) 2014;21:131-9.  Back to cited text no. 120
Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 1998;273:5858-68.  Back to cited text no. 121
Rogakou EP, Boon C, Redon C, Bonner WM. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol 1999;146:905-16.  Back to cited text no. 122
Burma S, Chen BP, Murphy M, Kurimasa A, Chen DJ. ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J Biol Chem 2001;276:42462-7.  Back to cited text no. 123
Stiff T, O'Driscoll M, Rief N, Iwabuchi K, Löbrich M, Jeggo PA, et al. ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizing radiation. Cancer Res 2004;64:2390-6.  Back to cited text no. 124
Stucki M, Clapperton JA, Mohammad D, Yaffe MB, Smerdon SJ, Jackson SP, et al. MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks. Cell 2005;123:1213-26.  Back to cited text no. 125
Livingstone M, Ruan H, Weiner J, Clauser KR, Strack P, Jin S, et al. Valosin-containing protein phosphorylation at ser784 in response to DNA damage. Cancer Res 2005;65:7533-40.  Back to cited text no. 126
Jiang N, Shen Y, Fei X, Sheng K, Sun P, Qiu Y, et al. Valosin-containing protein regulates the proteasome-mediated degradation of DNA-PKcs in glioma cells. Cell Death Dis 2013;4:e647.  Back to cited text no. 127
van den Boom J, Wolf M, Weimann L, Schulze N, Li F, Kaschani F, et al. VCP/p97 extracts sterically trapped ku70/80 rings from DNA in double-strand break repair. Mol Cell 2016;64:189-98.  Back to cited text no. 128
Jiang Y, Qian X, Shen J, Wang Y, Li X, Liu R, et al. Local generation of fumarate promotes DNA repair through inhibition of histone H3 demethylation. Nat Cell Biol 2015;17:1158-68.  Back to cited text no. 129
Farber-Katz SE, Dippold HC, Buschman MD, Peterman MC, Xing M, Noakes CJ, et al. DNA damage triggers golgi dispersal via DNA-PK and GOLPH 3. Cell 2014;156:413-27.  Back to cited text no. 130
Matsuoka S, Ballif BA, Smogorzewska A, McDonald ER 3rd, Hurov KE, Luo J, et al. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science 2007;316:1160-6.  Back to cited text no. 131
Kotula E, Faigle W, Berthault N, Dingli F, Loew D, Sun JS, et al. DNA-PK target identification reveals novel links between DNA repair signaling and cytoskeletal regulation. PLoS One 2013;8:e80313.  Back to cited text no. 132
Sishc BJ, Davis AJ. The role of the core non-homologous end joining factors in carcinogenesis and cancer. Cancers (Basel) 2017;9:81.  Back to cited text no. 133


  [Figure 1], [Figure 2], [Figure 3]


Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

  In this article
Historical Persp...
Structure of Ku ...
Components and M...
Role of Protein ...
Role of Protein ...
Concluding Remar...
Article Figures

 Article Access Statistics
    PDF Downloaded76    
    Comments [Add]    

Recommend this journal