DNA-dependent protein kinase (DNA-PK) is composed of a DNA-PK catalytic subunit (DNA-PKcs) and Ku heterodimer (hereafter denoted Ku), which consists of Ku80 (also termed Ku86) and Ku70 [4,5]. DNA-PK binds to and is activated by the end of a double-stranded DNA (dsDNA). Thus, DNA-PK acts as the sensor for the end of dsDNA, which appears when a DSB is generated.

DNA-PKcs is a huge protein consisting of 4128 amino acids (Figure 2A) [6]. DNA-PKcs is structurally related to Ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases, which are also implicated in DNA repair and DNA damage response [6,7,8,9]. ATM is recruited to DSB by the MRN complex consisting of Mre11, Rad50 and Nbs1, the last of which is responsible for Nijmegen breakage syndrome [10,11]. ATR is recruited to ssDNA through interaction with ATR-interacting protein (ATRIP) and Replication Protein A (RPA) [12]. These kinases show structural similarity to phosphatidylinositol 3-kinases (PI3Ks) and assemble the PIKK family. There are three additional PIKK members in humans, i.e., mammalian Target of rapamycin (mTOR, also termed FKBP12-rapamycin-associated protein, FRAP, and Rapamycin and FKBP12-target, RAFT) [13,14], Suppressor of morphological defects on genitalia-1 (SMG-1) [15,16], and Transformation/transcription domain-associated protein (TRRAP) [17]. Interestingly, TRRAP lacks kinase catalytic activity [17]. In addition to the kinase domain, these proteins share the FAT (FRAP, ATM and TRRAP), PRD (PIKK-regulatory domain) and FATC (FAT C-terminal) domains (Figure 2A). The primary function of mTOR is the regulation of cell growth and survival [13,14]. SMG-1 is essential for nonsense-mediated mRNA decay (NMD) [15,16]. PI3Ks mediates the signals from G-protein coupled receptors and receptor tyrosine kinases through the activation of AKT protein kinase (also known as protein kinase B, PKB) and mTOR [18,19,20]. This signaling pathway is called the PI3K/AKT/mTOR pathway. There are lines of evidence implicating these molecules in DNA damage response, although less directly than DNA-PKcs, ATM, and ATR [18,19,20]. SMG-1 was shown to activate the G1/S checkpoint through p53 upregulation and Cdc25A downregulation [21,22]. Recent studies showed that the human papilloma virus E6 protein and DNMT1 enhance radiosensitivity via the downregulation of SMG-1 [23,24]. The PI3K/AKT/mTOR pathway is upregulated in response to radiation and promotes cell survival [25]. AKT inhibits apoptosis through the downregulation of proapoptotic proteins, such as B cell lymphoma 2 associated agonist of cell death (BAD) [26,27] and upregulation of antiapoptotic proteins, such as human homolog of murine double minute 2 (HDM2), which promotes the degradation of p53 [28]. It is also reported that AKT augments NHEJ and HR [29,30]. Moreover, DNA-PK is shown to activate AKT in response to DNA damage directly or indirectly via Sty1/Spc1-interacting protein 1 (Sin1) [31,32]. The upregulation of the PI3K/AKT/mTOR pathway is frequently found in various types of cancer and is associated with resistance to radiotherapy and chemotherapy [18,19,20]. Thus, mTOR and PI3Ks are considered promising targets for radiosensitization and chemosensitization.

Ku80 and Ku70 consist of 732 and 609 amino acids, respectively (Figure 2B) [33,34]. Ku was initially identified as the antigen against autoantibody in a patient with an autoimmune disease, scleroderma-polymyositis overlap syndrome. Ku binds to the end of dsDNA without any particular preference in the nucleotide sequence [35]. DNA-PKcs is recruited to the end of dsDNA via interaction with the C-terminal region of Ku80 [36].

X-ray crystallography showed that Ku forms a ring-shaped structure that can encircle DNA, accounting for how Ku binds selectively to DNA ends [37]. Recent cryoelectron microscopy (cryo-EM) studies revealed a structure of DNA-PKcs complexed with Ku and DNA [38] (Figure 2C). DNA-PKcs is folded into a ring and a head. The ring includes the HEAT repeats and the interface with Ku, while the head includes FAT, kinase, PRD, and FAT-C domains [38]. DNA is inserted into the rings of Ku and DNA-PKcs [38]. The structural differences between inactive and active states of DNA-PKcs are also revealed. Most notably, PRD is closed in the inactive state and is assumed to clash with the substrate polypeptide. However, PRD becomes open in the active state, allowing the entry of a substrate polypeptide. Another cryo-EM study revealed the structure of DNA-PKcs in a complex with adenosine-5′-(γ-thio)-triphosphate (ATPγS), which is a non-hydrolyzable ATP analog [39] (Figure 2D). The adenine group is inserted into a hydrophobic pocket surrounded by Tyr3791, Trp3805 and Leu3806 [39]. Three phosphate groups and Mg²⁺ ions come in contact with Asn3926, Asn3927, Asp3941, Ser3731 and Lys3753 [39]. These multiple interactions are thought to stabilize the interaction between DNA-PKcs and ATP. While Lys3753, Tyr3791, Trp3805, Asn3927 and Asp3941 are fully conserved among PIKKs, Ser3731, Leu3806 and Asn3926 are divergent. The structures of DNA-PKcs with inhibitors were also elucidated (see below).

Ku and DNA-PKcs have been shown to be essential for NHEJ (Figure 2E and for details, refer to another review [40]). Initially, Ku80 was shown to correspond to X-ray repair cross-complementing group (XRCC) 5, which is deficient in a series of rodent cell lines exhibiting hypersensitivity to IR and defective V(D)J recombination [41,42]. Subsequently, DNA-PKcs was shown to correspond to XRCC7 and to be the responsible gene for murine severe combined immunodeficiency (scid) mutation [43,44,45]. Thereafter, a number of cells and animals deficient for DNA-PKcs, Ku80 or Ku70 were found or generated through gene targeting or genome editing [40]. In addition, six human individuals that harbor homozygous or compound heterozygous mutations in DNA-PKcs have been identified [40].

NHEJ proceeds in three stages (Figure 2E). In the recognition stage, Ku first binds to the end of DNA and then recruits DNA-PKcs. Paralog of XRCC4 and XLF (PAXX) stabilizes the binding of Ku to DNA and facilitates the subsequent assembly of the NHEJ factors [45,46,47,48]. When DNA ends are not compatible, they undergo the processing stage (for details, refer to review [2]). Artemis, in a complex with DNA-PKcs, exerts endonuclease activity on hairpin and overhang structures and 5′ to 3′ exonuclease activity on single-stranded DNA [49,50]. DNA polymerase μ (Polμ) and DNA polymerase λ (Polλ) fill in the gaps in DSBs. Polynucleotide kinase phosphatase (PNKP) adds a phosphate group at the 5′-end if absent and removes the phosphate group present at the 3′-end. Aprataxin (APTX) removes adenosinemonophosphate (AMP) from the abortive intermediates of ligation. Tyrosyl-DNA phosphodiesterase 1 (TDP1) and TDP2 remove the covalently bound proteins and phosphoglycolate groups from 3′-ends. In the ligation stage, DNA ligase IV (LIG4), which is associated with XRCC4, joins two DNA ends together [51,52,53]. XRCC4-like factor (XLF, also known as Cernunnos) forms filaments with XRCC4, which are suggested to align or bridge two DNA ends [54,55,56].

The kinase activity of DNA-PKcs is required for NHEJ because the catalytically inactive (kinase-dead) form of DNA-PKcs cannot rescue the radiosensitivity and V(D)J recombination defects of DNA-PKcs-deficient cells [57,58]. Although the precise roles of protein phosphorylation by DNA-PKcs remain elusive, DNA-PKcs is shown to phosphorylate NHEJ factors and other potentially NHEJ-related proteins (Table 1). The significance of phosphorylation of each substrate protein has been discussed elsewhere [59]. A number of small molecules that inhibit DNA-PK have been developed to date. These compounds have been powerful tools to delineate the function of DNA-PK. Furthermore, they are promising agents in cancer therapy, sensitizing cancer cells to radiotherapy and chemotherapy.

References

1. United Nations Scientific Committee on the Effects of Atomic Radiation. Report of the United Nations Scientific Committee on the Effects of Atomic Radiation 2010 Fifty-seventh session, includes Scientific Report: summary of low-dose radiation effects on health.
2. Zhao, B.; Rothenberg, E.; Ramsden, D.A.; Lieber, M.R. The molecular basis and disease relevance of non-homologous DNA end joining. Nat Rev Cell Biol. 2020, 21, 765–781.
3. Yilmaz, D.; Furst, A.; Meaburn, K.; Lezaja, A.; Wen, Y.; Altmeyer, M.; Reina-San-Martin, B.; Soutoglou, E. Activation of homologous recombination in G1 preserves centromeric integrity. Nature 2021, 600, 748–753.
4. Dvir, A.; Stein, L.Y.; Calore, B.L.; Dynan, W.S. Purification and characterization of a template-associated protein kinase that phosphorylates RNA polymerase II. Biol. Chem. 1993, 268, 10440–10447.
5. Gottlieb, T.M.; Jackson, S.P. The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell 1993, 72, 131–142.
6. Hartley, K.; Gell, D.; Smith, C.; Zhang, H.; Divecha, N.; Connelly, M.; Admon, A.; Lees-Miller, S.; Anderson, C.; Jackson, S. DNA-dependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product. Cell 1995, 82, 849–856.
7. Savitsky, K.; Bar-Shira, A.; Gilad, S.; Rotman, G.; Ziv, Y.; Vanagaite, L.; Tagle, D.A.; Smith, S.; Uziel, T.; Sfez, S. et al. A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science 1995, 268, 1749–1753.
8. Bentley, N.J.; Holtzman, D.A.; Flaggs, G.; Keegan, K.S.; DeMaggio, A.; Ford, J.C.; Hoekstra, M.; Carr, A.M. The Schizosaccharomyces pombe rad3 checkpoint gene. EMBO J. 1996, 15, 6641–6651.
9. Cimprich, K.A.; Shin, T.B.; Keith, C.T.; Schleiber, S.L. cDNA cloning and gene mapping of a candidate human cell cycle checkpoint protein. Natl. Acad. Sci. USA 1996, 93, 2850–2855.
10. Girard, P.M.; Riballo, E.; Begg, A.C.; Waugh, A.; Jeggo P.A. Nbs1 promotes ATM dependent phosphorylation events including those required for G1/S arrest. Oncogene 2002, 21, 4191-4199.
11. Uziel, T.; Lerenthal, Y.; Moyal, L.; Andegeko, Y.; Mittelman, L.; Shiloh, Y. Requirement of the MRN complex for ATM activation by DNA damage. EMBO J. 2003, 22, 5612–5621.
12. Zou, L.; Elledge, S.J. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science. 2003, 300, 1542–1548.
13. Brown, E.J.; Albers, M.W.; Shin, T.B.; Ichikawa, K.; Keith, C.T.; Lane, W.S.; Schreiber, S.L. A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature 1994, 369, 756–758.
14. Sabatini, D.M.; Erdjument-Bromage, H.; Lui, M.; Tempst, P.; Snyder, S.H. RAFT1: A mammalian protein that binds to FKBP12 in a Rapamycin-dependent fashion and is homologous to yeast TORs. Cell 1994, 78, 35–43.
15. Denning, G.; Jamieson, L.; Maquat, L.E.; Thompson, E.A.; Fields, A.P. Cloning of a novel phosphatidylinositol kinase-related kinase: characterization of the human SMG-1 RNA surveillance protein. Biol. Chem. 2001, 276, 22709–22714.
16. 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, 2215–2228.
17. McMahon, S.B.; Van Buskirk, H.A.; Dugan, K.A.; Copeland, T.D.; Cole, M.D. The novel ATM-related protein TRRAP is an essential cofactor for the c-Myc and E2F oncoproteins. Cell 1998, 94, 363–374.
18. Alemi, F.; Sadigh, A.R.; Malakoti, F.; Elhaei, Y.; Ghaffari, S.H.; Maleki, M.; Asemi, Z.; Yousefi, B.; Targhazeh, N.; Majidinia, M. Molecular mechanisms involved in DNA repair in human cancers: An overview of PI3k/Akt signaling and PIKKs crosstalk. Cell Phisiol. 2022, 237, 313–328.
19. Huang, T.T.; Lampert, E.J.; Coots, C.; Lee, J.M. Targeting the PI3K pathway and DNA damage response as a therapeutic strategy in ovarian cancer. Cancer Treat. Rev. 2020, 86, 102021.
20. Wanigasooriya, K.; Tyler, R.; Barros-Silva, J.D.; Sinha, Y.; Ismail, T.; Beggs, A.D. Radiosensitising Cancer Using Phosphatidylinositol-3-Kinase (PI3K), Protein Kinase B (AKT) or Mammalian Target of Rapamycin (mTOR) Inhibitors. Cancers (Basel) 2020, 12, 1278.
21. Gewandter, J.S.; Bambara, R,A.; O’Reilly, M.A. The RNA surveillance protein SMG1 activates p53 in response to DNA double-strand breaks but not exogenously oxidized mRNA. Cell Cycle 2011, 10, 2561–2567.
22. Gubanova, E.; Issaeva, N.; Gokturk, C.; Djureinovic, T.; Helleday, T. SMG-1 suppresses CDK2 and tumor growth by regulating both the p53 and Cdc25A signaling pathways. Cell Cycle 2013, 12, 3770–3780.
23. Zhang, C.; Mi, J.; Deng, Y.; Deng, Z.; Long, D.; Liu, Z. DNMT1 Enhances the Radiosensitivity of HPV-Positive Head and Neck Squamous Cell Carcinomas via Downregulating SMG1. Targets Ther. 2020, 13, 4201–4211.
24. Long, D.; Xu, L.; Deng, Z.; Guo, D.; Zhang, Y.; Liu, Z.; Zhang, C. HPV16 E6 enhances the radiosensitivity in HPV-positive human head and neck squamous cell carcinoma by regulating the miR-27a-3p/SMG1 axis. Agent Cancer 2021, 16, 56.
25. Chen, Y.H.; Wei, M.F.; Wang, C.W.; Lee, H.W.; Pan, S.L.; Gao, M.; Kuo, S.H.; Cheng, A.L.; Teng, C.M. Dual phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor is an effective radiosensitizer for colorectal cancer. Cancer Lett. 2015, 357, 582–590.
26. del Peso, L.; González-García, M.; Page, C.; Herrera, R.; Nuñez, G. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science1997, 278, 687–689.
27. Datta, S.R.; Dudek, H.; Tao, X.; Masters, S.; Fu, H.; Gotoh, Y.; Greenberg, M.E. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 1997, 91, 231–241.
28. Zhou, B.P.; Liao, Y.; Xia, W.; Zou, Y.; Spohn, B.; Hung, M.C. HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation. Cell Biol. 2001, 3, 973–982.
29. Oeck, S.; Al-Refae, K.; Riffkin, H.; Wiel, G.; Handrick, R.; Klein, D.; Iliakis, G.; Jendrossek, V. Activating Akt1 mutations alter DNA double strand break repair and radiosensitivity. Rep. 2017, 7, 42700.
30. Mueck, K.; Rebholz, S.; Harati, M.D.; Rodemann, H.P.; Toulany, M. Akt1 Stimulates Homologous Recombination Repair of DNA Double-Strand Breaks in a Rad51-Dependent Manner. J. Mol. Sci. 2017, 18, 2473.
31. Bozulic, L.; Surucu, B.; Hynx, D.; Hemmings, B.A. PKBalpha/Akt1 acts downstream of DNA-PK in the DNA double-strand break response and promotes survival. Cell 2008, 30, 203–213.
32. Liu, L.; Dai, X.; Yin, S.; Liu, P.; Hill, E.G.; Wei, W.; Gan, W. DNA-PK promotes activation of the survival kinase AKT in response to DNA damage through an mTORC2-ECT2 pathway. Signal 2022, 15, eabh2290.
33. Yaneva, M.; Wen, J.; Ayala, A.; Cook, R. cDNA-derived amino acid sequence of the 86-kDa subunit of the Ku antigen. Biol. Chem. 1989, 264, 13407–13411.
34. Reeves, W.H.; Sthoeger, Z.M. Molecular cloning of cDNA encoding the p70 (Ku) lupus autoantigen. Biol. Chem. 1989, 264, 5047–5052.
35. Mimori, T.; Hardin, J.A. Mechanism of interaction between Ku protein and DNA. Biol. Chem. 1986, 261, 10375–10379.
36. Falck, J.; Coates, J.; Jackson, S.P. Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage. Nature 2005, 434, 605–611.
37. Walker, J.R.; Corpina, R.A.; Goldberg, J. Structure of the Ku heterodimer bound to DNA and its implication for double-strand break repair. Nature 2001, 412, 607–614.
38. Chen X, Xu X, Chen Y, Cheung JC, Wang H, Jiang J, de Val N, Fox T, Gellert M, Yang W. Structure of an activated DNA-PK and its implications for NHEJ. Cell 2021, 81, 801–810.
39. Liang, S.; Thomas, S.E.; Chaplin, A.K.; Hardwick, S.W.; Chirgadze, D.Y.; Blundell, T.L. Structural insights into inhibitor regulation of the DNA repair protein DNA-PKcs. Nature 2022, 601, 643–648.
40. Matsumoto, Y.; Asa A.D.D.C..; Modak, C.; Shimada M. DNA-dependent protein kinase catalytic subunit: the sensor for DNA double-strand breaks structurally and functionally related to Ataxia telangiectasia mutated. Genes 2021, 12, 1143.
41. Taccioli, G.E.; Gottlieb, T.M.; Blunt, T.; Priestley, A.; Demengeot, J.; Mizuta, R.; Lehmann, A.R.; Alt, F.W.; Jackson, S.P.; Jeggo, P.A. Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination. Science 1994, 265, 1442–1445.
42. Smider, V.; Rathmell, W.K.; Lieber, M.R.; Chu, G. Restoration of X-ray resistance and V(D)J recombination in mutant cells by Ku cDNA. Science 1994, 266, 288–291.
43. Blunt, T.; Finnie, N.J.; Taccioli, G.E.; Smith, G.C.; Demengeot, J.; Gottlieb, T.M.; Mizuta, R.; Varghese, A.J.; Alt, F.W.; Jeggo, P.A. 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, 813–823.
44. Kirchgessner, C.U.; Patil, C.K.; Evans, J.W.; Cuomo, C.A.; Fried, L.M.; Carter, T.; Oettinger, M.A.; Brown, J.M. DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect. Science 1995, 267, 1178–1183.
45. Peterson, S.R.; Kurimasa, A.; Oshimura, M.; Dynan, W.S.; Bradbury, E.M.; Chen, D.J. Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells. Natl. Acad. Sci. USA 1995, 92, 3171–3174.
46. Ochi, T.; Blackford, A.N.; Coates, J.; Jhujh, S.; Mehmood, S.; Tamura, N.; Travers, J.; Wu, Q.; Draviam, V.M.; Robinson, C.V.; et al. DNA repair. PAXX, a paralog of XRCC4 and XLF, interacts with Ku to promote DNA double-strand break repair. Science 2015, 347, 185–188.
47. Xing, M.; Yang, M.; Huo, W.; Feng, F.; Wei, L.; Jiang, W.; Ning, S.; Yan, Z.; Li, W.; Wang, Q.; et al. Interactome analysis identifies a new paralogue of XRCC4 in non-homologous end joining DNA repair pathway. Commun. 2015, 6, 6233.
48. Craxton, A.; Somers, J.; Munnur, D.; Jukes-Jones, R.; Cain, K.; Malewicz, M. XLS (c9orf142) is a new component of mammalian DNA double-stranded break repair. Cell Death Diff. 2015, 22, 890–897.
49. Moshous, D.; Callebaut, I.; de Chasseval, R.; Corneo, B.; Cavazzana-Calvo, M.; Le Deist, F.; Tezcan, I.; Sanal, O.; Bertrand Y.; Philippe, N.; 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, 177–186.
50. Ma, Y.; Pannicke, U.; Schwarz, K.; Lieber, M.R. 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, 781–794.
51. Li, Z.; Otevrel, T.; Gao, Y.; Cheng, H.; Seed, B.; Stamato, T.; Taccioli, G.E.; Alt, F.W. The XRCC4 gene encodes a novel protein involved in DNA double-strand break repair and V(D)J recombination. Cell 1995, 83, 1079–1089.
52. Critchlow, S.; Bowater, R.; Jackson, S. Mammalian DNA double-strand break repair protein XRCC4 interacts with DNA ligase IV. Biol. 1997, 7, 588–598.
53. Grawunder, U.; Wilm, M.; Wu, X.; Kulesza, P.; Wilson, T.; Mann, M.; Michael, R.; Leiber. Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells. Nature 1997, 388, 492–495.
54. Buck, D.; Malivert, L.; de Chasseval, R.; Barraud, A.; Fondanèche, M.C.; Sanal, O.; Plebani, A.; Stéphan, J.L.; Hufnagel, M.; le Deist, F. et al. Cernunnos.; a novel nonhomologous end-joining factor.; is mutated in human immunodeficiency with microcephaly. Cell 2006, 124, 287–299.
55. Ahnesorg, P.; Smith, P.; Jackson, S.P. XLF interacts with the XRCC4-DNA ligase IV complex to promote DNA nonhomologous end-joining. Cell 2006, 124, 301–313.
56. Hammel, M.; Yu, Y.; Fang, S.; Lees-Miller, S.P.; Tainer, J.A. XLF regulates filament architecture of the XRCC4·ligase IV complex. Structure 2010, 18, 1431–1442.
57. Kienker, L.J.; Shin, E.K.; 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, 2752–2761.
58. Kurimasa, A.; Kumano, S.; Boubnov, N.; Story, M.; Tung, C.; Peterson, S.; Chen, D.J. Requirement for the kinase activity of human DNA-dependent protein kinase catalytic subunit in DNA strand break rejoining. Cell Biol. 1999, 19, 3877–3884.
59. Matsumoto, Y.; Sharma, M.K. DNA-dependent protein kinase in DNA damage response: Three decades and beyond. Radiat. Cancer Res. 2020, 11, 123–134.
60. Sehnal D, Bittrich S, Deshpande M, Svobodová R, Berka K, Bazgier V, Velankar S, Burley SK, Koča J, Rose AS. Mol* Viewer: modern web app for 3D visualization and analysis of large biomolecular structures. Nucleic Acids Res. 2021, 49, 431–437.