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 Table of Contents  
REVIEW ARTICLE
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
Japan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jrcr.jrcr_60_20

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  Abstract 


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 Jan 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]

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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

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  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

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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

1.
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