The Ku heterodimer: Function in DNA repair and beyond

Mutation Research 763 (2015) 15–29

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Review

The Ku heterodimer: Function in DNA repair and beyond Victoria L. Fell, Caroline Schild-Poulter * Robarts Research Institute and Department of Biochemistry, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario N6A 5B7, Canada

A R T I C L E I N F O

A B S T R A C T

Article history: Received 11 April 2014 Received in revised form 7 June 2014 Accepted 25 June 2014 Available online 4 July 2014

Ku is an abundant, highly conserved DNA binding protein found in both prokaryotes and eukaryotes that plays essential roles in the maintenance of genome integrity. In eukaryotes, Ku is a heterodimer comprised of two subunits, Ku70 and Ku80, that is best characterized for its central role as the initial DNA end binding factor in the ‘‘classical’’ non-homologous end joining (C-NHEJ) pathway, the main DNA double-strand break (DSB) repair pathway in mammals. Ku binds double-stranded DNA ends with high affinity in a sequence-independent manner through a central ring formed by the intertwined strands of the Ku70 and Ku80 subunits. At the break, Ku directly and indirectly interacts with several C-NHEJ factors and processing enzymes, serving as the scaffold for the entire DNA repair complex. There is also evidence that Ku is involved in signaling to the DNA damage response (DDR) machinery to modulate the activation of cell cycle checkpoints and the activation of apoptosis. Interestingly, Ku is also associated with telomeres, where, paradoxically to its DNA end-joining functions, it protects the telomere ends from being recognized as DSBs, thereby preventing their recombination and degradation. Ku, together with the silent information regulator (Sir) complex is also required for transcriptional silencing through telomere position effect (TPE). How Ku associates with telomeres, whether it is through direct DNA binding, or through protein–protein interactions with other telomere bound factors remains to be determined. Ku is central to the protection of organisms through its participation in C-NHEJ to repair DSBs generated during V(D)J recombination, a process that is indispensable for the establishment of the immune response. Ku also functions to prevent tumorigenesis and senescence since Ku-deficient mice show increased cancer incidence and early onset of aging. Overall, Ku function is critical to the maintenance of genomic integrity and to proper cellular and organismal development. ß 2014 Published by Elsevier B.V.

Keywords: Non-homologous end joining Ku Telomere DNA repair Cancer Aging

Contents 1. 2. 3.

4. 5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ku structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ku in DNA repair . . . . . . . . . . . . . . . . . . . . . . . . . . Non-homologous end joining . . . . . . . . . . . 3.1. Ku removal . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Competition between repair pathways . . . 3.3. Role of Ku in other DNA repair pathways . 3.4. Ku as a DNA damage signaling molecule . . . . . . . Ku at telomeres . . . . . . . . . . . . . . . . . . . . . . . . . . . Ku in disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immune system disorders . . . . . . . . . . . . . 6.1. 6.2. Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.

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* Corresponding author at: Robarts Research Institute, Western University, 115 Richmond Street North, London, Ontario N6A 5B7, Canada. Tel.: +1 519 931 5777x24164; fax: +1 519 931 5252. E-mail address: [email protected] (C. Schild-Poulter). http://dx.doi.org/10.1016/j.mrrev.2014.06.002 1383-5742/ß 2014 Published by Elsevier B.V.

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7.

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Ku was first identified in the early 1980s as an autoantigen targeted by autoantibodies in the serum of patients diagnosed with an autoimmune disease known as scleroderma polymyositis overlap syndrome [1]. The name Ku comes from the first two letters of the name of the original patient in whose serum it was identified. Autoantibodies directed against Ku were subsequently found in several other autoimmune diseases, including systemic lupus erythematosus, Sjorgren’s syndrome, polymyositis and scleroderma [2–4]. Early studies using serum from Ku-positive patients identified Ku as an abundant, mostly nuclear protein [1,5]. Subsequent reports showed that Ku had unusual DNA binding properties, binding avidly to the ends of double-stranded DNA molecules in a sequence-independent manner, and to a lesser extent, to other forms of DNA discontinuities such as hairpins gaps and nicks [5–7]. These unusual end-binding properties made Ku an appealing candidate for a role in DSB

A

23 24 24

repair and V(D)J recombination, which was confirmed when the two Ku subunits (XRCC5, Ku80 and XRCC6, Ku70) were found to complement the DNA repair defect of several IR-sensitive cell lines [8–14]. It is now well established that while not essential to individual life in the short term, Ku function is critical to the maintenance of genomic integrity and to the proper cellular and organismal development. A better understanding of Ku’s diverse roles at the cellular and organismal level have implications for the study and the treatment of other human diseases, such as immune system disorders, cancer and aging. 2. Ku structure Ku is a highly abundant protein found in vivo as a stable heterodimer consisting of two subunits, Ku70 and Ku80 (70 and 80 kDa, respectively). Both Ku70 and Ku80 eukaryotic Ku subunits contain three domains (Fig. 1A): an N-terminal alpha helix/beta barrel von Willebrand A (vWA) domain; a central core domain

Ku70 P

P

Human

P

AcAc Ac

vWa

AcAc AcAc

SAP

DNA binding core NLS

Yeast

Ku80 P P

DNA binding core

vWa

Human

DNA-PK binding P

CTD NLS

Yeast

Prokaryotes

B

DNA binding core

Ku80 vWA

Ku70 vWA

Ku70 SAP Fig. 1. Representation of Ku structure. (A) Schematic diagram of domain representation of the Ku70 and Ku80 subunits. The subunit domain structure of yeast and human Ku consists of the alpha helix, beta barrel N-terminal vWA domain, a central DNA binding core and a C-terminal helical domain (CTD). The eukaryotic Ku80 CTD contains the region required for binding DNA-PKCS, while the Ku70 CTD is shorter and contains a SAF-A/B, Acinus and PIAS domain (SAP). The location of the nuclear localization signals and post translation modifications (phosphorylation and acetylation) on the human Ku protein is indicated. Yeast Ku is comprised of a similar domain structure to human Ku, except for a truncated C-terminal domain in Ku80. Prokaryotes encode for a single Ku subunit that is homologous to the eukaryotic core DNA binding domain. (B) The crystal structure of human Ku (PDB: IJEY) colored according to the domain structure. The dimer forms an asymmetrical basket structure with a positively charged ring large enough to accommodate two turns of the DNA. The Ku80 C-terminus is not included in this crystal structure.

V.L. Fell, C. Schild-Poulter / Mutation Research 763 (2015) 15–29

required for DNA binding and dimerization; and a helical Cterminal domain. The Ku70/80 crystal structure (Fig. 1B) shows that the two subunits dimerize through the central domain to form a ring capable of accommodating two turns of double-stranded DNA (approximately 14 base pairs) [15]. This ring, consisting of intertwined strands of both Ku70 and Ku80, is lined with positively charged residues positioned to interact with the sugar phosphate backbone of DNA in a sequence-independent manner. Ku binds double-stranded DNA ends with high affinity (Kd  10–9 M), including 50 -30 or 30 -50 overhangs and blunt ends, however has significantly less affinity for circular DNA and single-stranded DNA ends [5,16,17]. Ku has a preferred orientation when loaded onto the DNA, placing Ku70 proximal to the DNA end, and Ku80 on the distal side, facing away from the end [15]. The N-terminal vWA domain (also called a/b domain) consists of a six-stranded beta-sheet in a Rossman fold. The amino side of the beta-sheet makes contact with the DNA groove, although it contributes little to Ku70/80 dimerization or DNA binding. The carboxyl end of the fold points away from the break, which makes it available as a protein–protein interaction surface [15]. This domain, named for its prototype protein, the von Willebrand Factor type A, is present in mostly extracellular matrix proteins but also several intracellular proteins, where it facilitates protein– protein interactions with a wide variety of ligands [18]. Overall, the function of this domain is not well characterized but there is emerging evidence that it has an important role in DSB repair and telomere regulation. In Saccaromyces cerevisae, mutations in helix 5 of Ku80 abrogated telomere silencing, while mutations in helix 5 of Ku70 had a negative impact on DSB repair in yeast and mouse cells [19–21]. Specific residues in the Ku70 vWA domain were also shown to be critically implicated in conferring abasic site processing by Ku in vitro [22,23]. Additionally, another region of the human Ku70 vWA domain in helix 4 was found to regulate DNA damage signaling to apoptosis [21]. Consistent with its predicted role as a protein–protein interaction surface, the vWA domain has been shown to mediate an interaction with both the telomere complex component telomere repeat binding factor 2 (TRF2) and NHEJ factor aprataxin and PNKP like factor (APLF) [20,24]. Both Ku subunits contain a helical C-terminal domain (CTD), however this is the most divergent region of the two proteins. The Ku80 CTD is approximately 15 kDa and contains a helical and a disordered region. In vertebrates, the extreme Ku80 C-terminus is involved in the recruitment of DNA-dependent protein kinase catalytic subunit (DNA-PKCS) to DNA [25,26]. Lower eukaryotes, such as Saccharomyces cerevisiae and Aradopsis thaliana, encode for a smaller Ku80 protein, missing this DNA-PKCS binding region, which correlates with the fact that DNA-PKCS is not present in these organisms. The Ku70 CTD is composed of a highly flexible linker region followed by a structured 5 kDa helix–loop–helix region known as the SAP domain (named after SAF-A/B, Acinus and PIAS motifs) [27]. The SAP domain has putative DNA binding properties and has been shown to increase the overall DNA binding affinity of the heterodimer [28,29]. This C-terminal region is also subject to post translational modifications. Multiple C-terminal lysine residues are acetylated to regulate Ku interaction with proapoptotic proteins [30,31]. Acetylation and sumoylation of Ku70’s C-terminal tail were also implicated in modulating its recruitment to DNA damage sites and Ku’s DNA binding affinity, respectively [31,32]. The Ku70 SAP domain was also shown to mediate the recruitment of homeodomain proteins to DNA ends [33]. Furthermore, Ku is primarily a nuclear protein, and the CTD in both subunits contains the basic nuclear localization signal motif that regulates the heterodimer’s nuclear transport [34]. It is currently unclear whether Ku subunits can exist alone in a monomeric form, but there is strong evidence that Ku is an obligate

17

heterodimer. Cells derived from Ku70 deficient mice show very low expression of Ku80, and similarly, the expression of Ku70 is severely reduced in cells derived from Ku80 deficient mice [14,35,36]. This phenomenon is also observed in yeast strains null for either Ku subunit, and in fact, these strains are phenotypically identical to the double Ku subunit knockout strain [37]. This is likely due to the instability of each subunit in the absence of their interacting partner, as exogenous re-expression of the missing subunit restores the protein levels of its heterodimeric partner [36]. Overall, evidence suggests that the individual Ku subunits are unstable and require heterodimerization to function. The Ku subunits share little primary sequence, but do have a conserved secondary structure, indicating that they may be derived from a common ancestor. Ku is an evolutionarily conserved protein, found in both the prokaryotic and eukaryotic kingdoms, and its overall structure is preserved throughout. As several homologues have been identified in bacterial and archaea species, Ku is thought to have prokaryotic origins [38,39]. For the most part, eukaryotic genomes contain a single copy of a Ku-like protein which also functions in a bacterial NHEJ pathway, but as a homodimer [40]. While having little DNA or protein sequence similarity to its eukaryotic counterparts, prokaryotic Ku homologues display structural homologies to the central core DNA binding domain region of both eukaryotic Ku subunits and form homodimers that bind double-stranded DNA ends [41]. Prokaryotic Ku is much smaller however, only approximately 30–40 kDa, lacking both the vWA and C-terminal domains found in eukaryotes [38,39]. 3. Ku in DNA repair 3.1. Non-homologous end joining There are three main DSB repair pathways in eukaryotes (Fig. 2): the classical NHEJ (C-NHEJ), alternative NHEJ or microhomology-mediated end joining (A-NHEJ or MMEJ) and homologous recombination (HR)[42–44]. C-NHEJ and A-NHEJ have the potential to be active in all stages of the cell cycle, however HR, is only active in the S and G2 phases because it utilizes the complementarity of the sister chromatid to repair the DSB with high accuracy [43,44]. C-NHEJ is capable of ligating any two DNA ends, regardless of sequence. Due to this flexibility, C-NHEJ is the predominant DSB repair pathway in humans and other higher eukaryotes [45–47]. C-NHEJ can be divided into five main stages: (I) recognition of the break by Ku, (II) Synaptic end bridging, (III) DNA end processing to produce compatible ends, (IV) ligation of the break, and finally, (V) Ku removal from the restored DNA. The importance of Ku to DSB repair has been well documented with reports of Ku deficient cells displaying inaccurate end-joining, dramatic radiosensitivity, and chromosomal breakage, translocations and aneuploidy [14,48–52]. The initial step of C-NHEJ is the rapid recognition of the DSB by Ku. Ku’s abundance (in humans, approximately 500,000 molecules per cell [5,15,53]) and strong affinity for DNA allows it to associate with DNA ends within 5 s of damage [54]. Ku binding has a protective role and in Ku-deficient cells, nucleolytic processing occurs at DSB ends [55]. Once Ku has encircled the DNA via its central ring domain, it directly and indirectly interacts with several NHEJ factors, serving as the scaffold for the entire NHEJ complex. Some uncertainty remains as to how many Ku molecules are recruited to a DNA break. Many DNA damage response factors form distinct foci easily visible at the site of DNA damage. However there has been difficulty in observing Ku following DNA damage by microscopy, leading to the conclusion that Ku does not accumulate in large numbers at a DSB. Furthermore, an in vitro study showed

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A

Homologous Recombination

B

Alternative Non Homologous End Joining

MRN

Region of microhomology

RPA Rad51 Rad54

BRCA2

PARP1

XRCC1

Rad52 Polymerase β

Ligase I/III

C

Classical Non Homologous End Joining DSB End Recognition

(1)

Ligation

(4)

Ku70 Ku80 Ligase IV

XRCC4

XLF

Ku80 Ku70 DNA PKcs

(2)

Synaptic End Joining P P

(5)

Ku Removal Artemis ? P P

Ub Ub Ub

Processing (3) Pol μ/λ

WRN

PNK

APLF

Protease

Fig. 2. Schematics of the double-strand break repair pathways in higher eukaryotes. Homologous recombination (HR) and classical non-homologous end joining (C-NHEJ) are the two main DSB repair pathways. HR employs a template sequence from a sister chromatid or a homologous chromosome to perform accurate repair of DSBs, and is restricted to the S and G2 phases of the cell cycle. HR is also involved in the resolution of stalled replication forks and mitotic and meiotic recombination [44]. C-NHEJ directly joins the two ends of a DSB, regardless of the sequence, and is a simple and rapid process available at any time during the cell cycle [45,46]. C-NHEJ is also involved in the immunoglobulin gene rearrangements of immune cells through V(D)J recombination and class-switch recombination (CSR)[193]. A third pathway is called by a variety of names: alternative NHEJ (alt NHEJ or A-NHEJ), microhomology-mediated end joining (MMEJ), and B (backup)-NHEJ. A-NHEJ is a highly error-prone pathway functioning in case of deficiencies in the C-NHEJ pathway [42]. (A) Homologous Recombination. After the introduction of a DSB, the ends are recognized by the MRN complex (Mre11-Rad50NBS1), which recruits proteins Sae2, Sgs1, Exo1 and Dna2 to facilitate resection of the break in the 50 direction and leave 30 single-stranded extensions. The 30 single-stranded overhangs are bound by the proteins RPA, Rad51, and Rad54 to form a nucleoprotein filament. The next step, aided by the recruitment of Rad52, is the invasion of this filament strand into the DNA of the homologous chromosome to form a D-loop structure. A polymerase extends the 30 overhang to create a cross structure known as the Holliday

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that only one or two Ku molecules were able to load onto a chromatin substrate [56]. A recently developed method for visualizing Ku foci also demonstrated that on average there are only two Ku molecules present at a DSB in vivo, presumably one at each end of the DNA break [57]. Ku is the DNA binding subunit of the DNA-dependent protein kinase (DNA-PK) complex, which comprises Ku and DNA-PK catalytic subunit (DNA-PKCS), a member of the phosphoinositide 3kinase-related kinase (PIKK) family [58]. DNA-PKCS activity is very important for successful DNA repair, as loss of DNA-PKCS activity through mutations or knockout, strongly diminishes C-NHEJ efficiency and, at the organismal level, results in profound immunodeficiency [10]. The low-resolution electron microscopic structure of the DNA-PK complex indicates that Ku70/80 makes several contacts with DNA-PKCS, including the N-terminal and Cterminal regions of DNA-PKCS [59]. At DSBs, DNA-PKCS is recruited by Ku to form the active complex, leading to Ku translocation and the eventual activation of DNA-PKCS catalytic activity. This translocation allows DNA-PKCS to be positioned at the tip of break and mediates the synaptic joining of the two broken ends and the stabilization of the complex [60]. Another important consequence of DNA-PK kinase activation are 15 auto-phosphorylation events on DNA-PKCS [61–65]. Ku70 and Ku80 were shown to be phosphorylated by DNA-PK in vitro, however the importance of these events in C-NHEJ function is unclear, as mutation of these residues does not impact cell survival after ionizing radiation (IR) [66]. Similarly, DNA-PK phosphorylates several NHEJ factors in vitro such as DNA ligase IV, X-ray cross complementing protein 4 (XRCC4), XLF (XRCC4 like factor, also known as Cernunnos) and Artemis but again many of these were dispensable for NHEJ in vivo [63,67–69]. There is increasing evidence that, along with DNA-PKCS, Ku itself mediates the DNA end bridging and stabilization of the CNHEJ ligation complex. Several early observations lead to the conclusion that Ku functions to bridge DNA ends, including the in vitro joining of two radiolabelled DNA ends by recombinant Ku protein and the visualization of Ku dependent DNA fragment joining by electron and atomic force microscopy [70,71]. More recently, utilizing an experimental system designed to visualize DSBs introduced by restriction endonucleases in mammalian cells, it was observed that the loss of Ku80 resulted in increased distance between DNA ends [72]. Despite these observations, there has been little insight into the precise mechanism of Ku end bridging. A mutation in helix 5 of the Ku70 vWA domain, noted for its ability to impair DSB repair in both yeast and mammalian cells, was found to decrease multimerization of Ku proteins, leading to the speculation that this helix mediates DNA end bridging through the binding of two Ku70 molecules [19–21]. Between the initial break recognition by Ku and the final ligation of the break, there is considerable flexibility in the factors involved in repair. For example, IR is known to produce a variety of damage to the DNA, occasionally leaving non-ligatable 30 phosphate groups, 30 -phosphoglycolates, or 50 -hydroxyl groups. Therefore, a number of kinases/phosphatases (polynucleotide kinase/phosphatase (PNKP)), nucleases (Werner, Mre11, Artemis, ExoI), polymerases (DNA polymerases m and l), helicases (RECQ1), and phosphodiesterases (tyrosyl-DNA phosphodiesterase 1) may

19

Table 1 A list of proteins proposed to directly interact with Ku, classified according to cellular process. NHEJ and V(D)J

DNA-PKCS [228]

Polymerase m [229] APLF [24] TdT [231] Rag-1 [233]

Polymerase l [230]

BRCA1 [124] PARP-1 [237] RECQ1 [239]

CAF-1 [235] NBS1 [140] ABH2 [130] Mre11 [240]

Kub5-Hera [236] INHAT [31] MSH6 [238] BARD1 [241]

Telomere

TRF1 [183] TRF2 [184] TLC1 [166] TERT [168]

HP1a [242] Rap1 [186] Sir3/4 [175] Mps3 [177]

Apoptosis

BAX [139] p18-cyclin E [141] HDAC6 [142] SIRT1 [143]

PCAF [30] Clusterin [243]

Cell cycle

CDK9 [244]

Cyclin A/CDK1 [117]

Transcription and replication

Oct-1 [33] HOXC4 [33] Dlx2 [33] TOP2A [251]

REF1 [245] ESE-1 [247] Runx2 [249] Dlx5 [249]

XRCC4/Lig4 [76] XLF [78] WRN [232] DNA repair (other)

g-H2AX [234]

CBP [30]

HIV-IN [246] Tat [248] HSF-1 [250] NAA15 [249]

be required to produce compatible DNA ends [73]. In the case of DSBs induced by Topoisomerase II (TOP2) poisons, TOP2 remains covalently linked to via a phosphotyrosyl bond to the 50 terminus and requires a specific end-processing enzyme, tyrosyl DNA phosphodiesterase 2 (TDP2), that hydrolyses 50 -phosphotyrosyl bonds at TOP2-associated DSBs [74,75]. In many cases, recruitment of these factors to the DNA break is dependent on Ku, with some factors directly interacting with Ku (Table 1). Interestingly, Ku itself has been shown to have some enzymatic activity and may also participate in the processing of DNA ends. It was identified as a 50 -dRP/AP lyase that removes abasic sites by nicking DNA 30 of the abasic site via a mechanism involving a Schiff-base covalent intermediate. Several lysine residues in the Ku70 vWA domain catalyze this reaction, notably K160/164, which when mutated to alanine residues completely inhibit Ku’s ability to form a Schiff base [22,23]. Following end-processing, the two DNA ends are ligated back together. Ku also has an important role in the recruitment of the ligase complex, which is comprised of DNA ligase IV, XRCC4 and XLF. The complex requires Ku to be recruited to the break and forms a direct interaction with Ku, with DNA ligase IV and XLF interacting with the whole heterodimer and XRCC4 interacting with Ku70 specifically [54,76–79]. There is still great uncertainty regarding the stepwise recruitment of NHEJ factors following Ku binding, although this is not surprising given that different processing enzymes are required in different situations. Indeed there is also some evidence that high complexity DSBs (containing single-strand overhangs, base oxidation and abasic sites) are repaired with slow kinetics and are dependent on DNA-PKCS activity, while low complexity DSBs (without surrounding DNA

junction, which is later cut to resolve the chromosome. (B) Alternative end joining/microhomology-mediated end joining (A-NHEJ/MMEJ). The break is recognized by the MRN complex, PARP1 and XRCC1, which promote the nucleolytic degradation of DNA to reveal 5–25 bp regions of microhomology between the DNA strands. After the alignment of complementary regions, the gaps are filled in by polymerase b, and ligated by either ligase I or ligase III. (C) Non homologous end joining (C-NHEJ). (1) The ends are recognized by the Ku heterodimer which slides directly onto the break via its DNA binding ring. The PIKK member DNA-PKCS binds at the Ku80 C-terminus to create the DNA-PK complex. (2) DNA-PK autophosphorylates to create the catalytically active DNA-PK complex. Artemis is recruited to DNA-PK and phosphorylated. (3) Several enzymes, including polymerases (polymerase m or l), nucleases, kinases (polynucleotide kinase) are recruited to the break and remove damaged bases or single-strand overhangs to create compatible ends for ligation. (4) The ligation complex, consisting of Ligase IV, XRCC4, and XLF are recruited by Ku and ligate the two DNA ends. (5) It is unclear how the Ku dimer is removed from the ligated break. Possible mechanisms include degradation of Ku by proteases and the ubiquitin pathway, or physical nicking of the repaired DNA by nucleases to allow Ku to slide off.

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damage) do not require DNA-PKCS binding, and are efficiently ligated with only Ku and the ligation complex [80–83]. 3.2. Ku removal One of the outstanding questions in NHEJ is how Ku is removed from DNA following ligation of the DSB. Ku is rapidly recruited to the break, but is only found there transiently, as laser microirradiation studies found that Ku signal steadily depletes within the few hours following the initial damage, presumably being removed as DNA is repaired [54,84]. Ku differs from many other DNA binding proteins in that it binds DNA ends by encircling them through its ring domain, which would suggest that once the two DNA breaks are ligated, Ku is trapped on the linear repaired DNA. Furthermore, the crystal and electron microscopy (EM) structures do not indicate an obvious escape mechanism given that the vWA and central ring domain conformations of Ku remain unchanged whether or not bound to DNA [15,59]. One possible mechanism is that the removal of Ku from DNA occurs via a protein degradation pathway. Studies in Xenopus laevis have shown that polyubiquitination of Ku80 Lysine 48 leads to its degradation by the Skp1-Cul1-Fbxl12 (SCF) E3 ubiquitin ligase complex [85]. Similarly in human cells, Ku80 ubiquitination was found to be dependent on the E3 ubiquitin ligase RING finger protein 8 (RNF8) [86]. The depletion of RNF8 led to increased Ku80 retention at the break and decreased NHEJ efficiency, suggesting that removal of Ku80 by ubiquitin-mediated degradation is an important step in successful DSB repair [86]. Another proposed mechanism for Ku removal is the direct nicking of DNA to allow Ku escape. Evidence for this has emerged from yeast systems, with the HR complex MRX (Mre11Rad50-Xrs2) performing an endonucleolytic incision adjacent to the DNA end, followed by digestion of the DNA to allow Ku removal and then restoration of the DNA by MRX and Dna2 resection [87– 89]. Overall, protein degradation and direct DNA nicking are very different possible mechanisms for Ku removal and further work needs to be done to resolve this discrepancy. It should be noted that these results were obtained from different biological systems and could indicate that there is a divergence in Ku removal mechanisms between yeast and higher eukaryotes. 3.3. Competition between repair pathways There is much investigation regarding how organisms regulate the DSB repair pathway choice and increasing evidence suggests that Ku has an inhibitory effect on the other DSB pathways. Ku is one of the first proteins found at DSB regardless of cell cycle stage and cells will first attempt to repair DSBs by C-NHEJ if the ends are compatible [84,90–92]. HR is the preferred pathway in the S and G2 phases, and the initial end binding factors of HR, for example Mre11, antagonize Ku for DNA end binding. The binding of HR factors initiates DNA end resection to produce single-stranded DNA, which Ku does not have a strong affinity for, and promotes the completion of DSB repair by HR [44]. In yeast, Ku appears to outcompete HR factors in G1 phase, as the loss of Ku results in increased Mre11 recruitment and Exo1 mediated resection [55,93– 96]. Overexpression of Ku is even able to reduce recruitment of Mre11 in G2, when HR is the preferred DSB repair pathway [93]. The HR inhibitory effect appears to be specifically dependent upon Ku’s DNA end binding function, as deletion of other NHEJ factors, such as ligase IV, were not able to increase HR activity to the same extent [93]. Another study has implicated, not only Ku binding, but the kinase activity of DNA-PKCS in the DNA repair pathway choice, as initiated of HR in G2 depended upon DNA-PKCS autophosphorylation events [97]. Ku70 also antagonizes HR via the Fanconi Anemia (FA) and break-induced replication (BIR) repair pathways. The FA pathway repairs DNA interstrand cross-links (ICLs) in

cooperation with the HR pathway during replication [98]. Cell lines deficient in FA genes display increased sensitivity to DNA damaging agents that specifically create DNA cross-links and show a greater dependence on repair by NHEJ. The simultaneous deletion of Ku70 however, reverses this sensitivity, and repair is completed by HR once again [99]. Similarly, BIR is responsible for the repair of breaks that occur in S phase following a replication fork collapse [100]. This pathway is also dependent on HR, and yeast cells null for Mre11 activity are sensitive to agents that induce replication stress [101]. Several Ku70 mutants were identified that rescue this Mre11 loss, again suggesting a competitive interplay between Ku and Mre11 [101,102]. A-NHEJ is Ku-independent end joining, and while it is not currently fully characterized, it is considered to be a more mutagenic DSB pathway as it occasionally utilizes microhomologies far from the DSB which results in extended resection and deletions at the repair site [42,43,103,104]. Similar to C-NHEJ, this pathway is active in all phases of the cell cycle, however it was only identified after the deletion of essential C-NHEJ components, suggesting that this pathway is secondary to C-NHEJ [103,105,106]. It is still unclear why A-NHEJ may be selected over the less mutagenic C-NHEJ pathway, however A-NHEJ, similar to HR, often begins with resection to form SSBs, which would inhibit Ku binding and promote the binding of A-NHEJ factors, such as PARP-1[42,43,103,104]. Ku outcompetes the DNA binding factor PARP-1, and deletion of Ku results in increased repair by this pathway [107–109]. Furthermore the DNA-PK complex has an inhibitory effect on the enzymatic activity of PARP-1 [110]. Studies have indicated that human somatic cell lines with Ku80 deletions retain DSB repair capability, but show a shift toward the A-NHEJ pathway [111]. However Ku80-expressing cell lines with individual deletions of the other proteins required for C-NHEJ have a dramatic decrease in all DSB repair pathways [111]. These results suggest that the binding of Ku, or the entire DNA-PK complex to DNA has a dominant negative effect on the other DSB repair pathways if C-NHEJ cannot be completed. The DNA repair pathway choice is largely determined by cell cycle stage, with breaks in G1 phase repaired by end joining, and breaks in G2/S phases largely repaired by HR. Not surprisingly, members of the cell cycle machinery, namely the cyclin-dependent kinases (CDKs), have been implicated in regulating the activity of several DNA repair factors [112,113]. Several studies have shown, in both yeast and mammalian systems, that CDKs phosphorylate the HR factors CtBP-interacting protein (CtIP) and Dna2 in order to initiate the switch to recombination at the G1/S transition [114– 116]. There is some evidence that Ku could also be regulated by CDK phosphorylation. Cyclin A1/CDK2 was implicated in the regulation of NHEJ following radiation and was proposed to be a binding partner of Ku in vertebrates [117]. Furthermore, mass spectrometry studies revealed potential CDK phosphorylation sites on Ku [118,119]. However, the functional significance of Ku phosphorylation by CDKs has yet to be elucidated. While several potential CDK1 phosphorylation sites were identified in S. cerevisae Ku, mutation of these sites did not elicit a DNA repair defect [120]. Recent work has also identified the factors p53 binding protein 1 (53BP1) and breast cancer 1 (BRCA1) as essential regulators in the pathway choice in mammalian cells. 53BP1, along with RAP1 interacting factor (Rif1) and Pax transactivation domain-interacting protein (PTIP), promote NHEJ and negatively regulate resection in G1 phase [121,122]. BRCA1, along with CtBP-interacting protein (CtiP), promotes the removal of 53BP1 during the switch to HR at the G1/S transition [123]. It is currently unclear how Ku fits into this mechanism, however there is evidence that Ku associates with BRCA1 and that this interaction is important for successful NHEJ in G1 [124–126]. It is possible that this association is essential for the removal of Ku from breaks, with BRCA1 utilizing the exonuclease

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activity of the MRN (Mre11-Rad50-NBS1) complex in a nicking mechanism analogous to that proposed for yeast Ku removal. 3.4. Role of Ku in other DNA repair pathways In addition to its role in the repair of DSBs by C-NHEJ, Ku has been implicated in a number of other DNA repair pathways. Ku has been suggested to function in base excision repair (BER), which repairs DNA base damage, apurinic/apyrimidic (AP) sites and single-strand breaks (SSBs) [127]. Cells deficient in Ku70 and Ku80 were sensitive to reactive oxygen species (ROS) and alkylation damage producing agents that result in base lesions and SSBs [128]. Additionally, in vitro experiments demonstrated that Ku subunits bind AP sites and cell extracts from Ku deficient cells have decreased BER activity [128,129]. Furthermore, Ku has been shown to interact with ABH2, an enzyme involved DNA alkylation repair, potentially implicating it in this pathway as well [130]. 4. Ku as a DNA damage signaling molecule The DNA damage response (DDR) pathway (Fig. 3) is initiated by sensor proteins that form large complexes that accumulate in foci at the site of damage (reviewed in [131]). This complex formation promotes the activation of a phosphorylation cascade, and the modification of surrounding chromatin to allow DNA repair factors to access the break. These initial sensors include the MRN complex, 53BP1, BRCA1, as well as the serine/threonine (S/T) PIKK family members Ataxia Telangiectasia Mutated (ATM), and Ataxia telangiectasia and Rad3 related (ATR) [132]. The PIKK members are the main regulators of the DNA damage response initiated at the DSB and orchestrate many phosphorylation events at the site of DNA damage. ATM/ATR phosphorylation of p53 and the checkpoint kinases (Chk1/2) initiate further signaling cascades to trigger the activation of DNA damage cell cycle checkpoints leading to cell cycle arrest and the possible initiation of apoptosis [133,134]. There is some evidence that Ku is involved in the modulation of ATM activity following DNA damage. Ku appears to prevent ATMdependent ATR activation, perhaps acting as a signaling block to the HR pathway [135]. Furthermore, Ku80 null cells were shown to have increased S phase inhibition after DNA damage due to increased ATM activity [136]. We identified a specific residue in Ku70’s vWA domain, serine 155, that when mutated to alanine results in decreased DNA damage signaling and apoptotic activation after IR, despite having no impact on Ku’s ability to

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function in C-NHEJ [21]. These results suggest that in addition to its essential role in DNA repair, modification of Ku also signals to the ATM dependent DDR cascade to regulate cell fate after DNA damage. This is not entirely surprising as several other DNA repair factors (ex. Mre11), have functions in both DNA repair as well as the DNA damage signaling cascade [137]. It is possible that Ku also has a dual role in the DNA damage response, first promoting the repair by NHEJ, as well as relaying signals as to the completion, or lack thereof, of DNA repair. In addition to its impact on upstream DDR signaling, a number of studies have implicated Ku70 as a direct inhibitor of apoptosis, through the binding and inhibition of the pro-apoptotic factor Bax [138,139]. This interaction is proposed to be modulated by various mechanisms, including protein–protein interactions and posttranslational modification of Ku. Some examples of protein interactions include the binding of Ku70 to NBS1 and a cleavage product of cyclin E, both of which were suggested to inhibit the binding of Ku70 to Bax and promote apoptosis [140,141]. The acetylation of Ku70 is also suggested to negatively regulate the Ku70-Bax interaction. Ku70 is acetylated in the C-terminal linker domain on eight lysine residues between K539 and K556 and alanine substitution of these residues decreased Bax-dependent apoptotic activation [30]. This acetylation is mediated by CREBbinding protein (CBP) and P300/CBP-associated factor (PCAF) while deacetylation is controlled by histone deacetylase (HDAC) and sirtuin (SIRT) deacetylase enzymes [30,142,143]. There is some evidence that Ku may have deubiquitination activity, as it has been proposed to promote the deubuquitination of Bax, and another anti-apoptotic regulator of Bax, Mcl-1 [138,144]. Overall, there are several unanswered questions regarding the interaction with Ku70 and Bax. Many studies have suggested that this interaction occurs in the absence of Ku80, despite the fact that there is little evidence that the Ku subunits exist as monomers. Furthermore, Ku is predominantly a nuclear protein but Bax is largely cytoplasmic in its inactive form and then translocates to the mitochondria during apoptotic activation [145]. The regulatory mechanisms that would allow a subset of Ku70 monomers to remain in the cytoplasm bound to Bax remain to be elucidated. 5. Ku at telomeres Telomeres are the linear ends of chromosomes and therefore have the potential to be recognized as DSBs and processed by DSB repair pathways. Specific protein complexes, such as the shelterin

P ATM

ATM P

P P Rif1

MDC1 P H2AX

P P

H2AX

DNA-PK CS

P BRCA1

53BP1 P

P

H2AX

H2AX

P H2AX

Rad50 Ku80 Ku70 P p53

P

Cell cycle arrest

Chk2 Apoptosis

Fig. 3. Overview of DNA double-strand break (DSB) recognition and DNA Damage Response (DDR). After the introduction of a double-strand break (DSB), the broken DNA end is rapidly recognized by the Ku heterodimer and the MRN complex (Mre11/Rad50/NBS1). Next is the recruitment of the PIKK family members DNA-PKcs, bound to Ku, and ATM, which both phosphorylate the histone variant H2AX on its C-terminal residue Ser139 (to yield g-H2AX). The MRN complex promotes the activation of ATM molecules, which in turn further amplify H2AX phosphorylation and spread the g-H2AX signal far from the initial damage site. Several other factors are phosphorylated by ATM and recruited to the damaged site in large numbers, including MDC1, 53BP1, Rif1 and BRCA1. ATM also initiates a signaling cascade through the phosphorylation of p53 and Chk1/ 2 that results in broad transcriptional changes to induce cell cycle arrest or activate apoptosis if the damage is unable to be repaired [131].

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complex in mammals and the Rap1 and the CST (Cdc13–Stn1– Ten1) complexes in yeast have evolved to bind and form protective caps on the DNA ends to prevent access by the DNA repair complexes (Fig. 4) [146,147]. A role for Ku in the maintenance of telomeres in yeast has been long established. Foundational to this was the observation that deletion of either Ku subunit in S. cerevisae resulted in shortened telomeres and long G-tails compared to wildtype [148–150]. The requirement of Ku for proper telomere structure and maintenance appears to be separate from C-NHEJ, as DNA Ligase IV deficient strains, the ligase essential for completion of C-NHEJ, do not show any telomere defects [151].

A Sir2/3/4 Rad50

TLC1 RNA

Mre11 Xrs2

Telomerase

Rap1

(b)

Ku70 Ku80

(a)

B TIN1

Mre11

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(a) Ku70 Ku80

(b)

Ku70

Fig. 4. General structure of telomeres. In most eukaryotes telomeric DNA consists of extended tracts of G-rich repeat arrays. The G-rich strand forms a short 30 singlestrand protrusion called the G-overhang, which mediates the formation of a complex secondary structure called a t-loop. The t-loop structure allows the folding of the end of the chromosome to mask and protect the DNA end from degradation. (A) Structure of the yeast telomere. The MRX (Mre11/Rad50/Xrs2) and Sir/Rap1 complexes directly bind the telomeric DNA [252]. Ku could potentially (a) bind the DNA end via its DNA binding domain or (b) bind via a protein–protein interaction with telomeric complexes (such as Sir/Rap1). Ku has a role in recruiting telomerase, mediated by an interaction with the TLC1 RNA and Ku80. Ku interaction with the telomere may be indirect, requiring the heterotetramerization of two Ku molecules. (B) Structure of the mammalian telomere. The Shelterin (TRF1/TRF2/Tin1/Pot1/ TPP1) and MRN (Mre11/Rad50/Nbs1) complexes coat the telomeric DNA to protect from degradation [253]. Again there is controversy whether (a) Ku directly binds the telomeric DNA or (b) is retained through a protein interaction with components of the Shelterin complex (such as TRF1/TRF2).

Analysis of telomeres in Ku-deficient mice has produced conflicting results regarding telomere length, with reports of telomere shortening as well as lengthening [152–154]. Despite this, the same mice exhibit increased telomere end-to-end fusions and chromosomal aberrations [153,154]. Similarly, human cells with deleted Ku80 show telomere loss and abnormal telomere structure, overall indicating a clear role for Ku in proper telomere structure maintenance in mammals [155]. Similar to its role in protecting the DNA ends of DSBs in NHEJ, Ku protects telomere ends from recombination and degradation events [148–150,152–154]. Paradoxically, although Ku promotes the fusion of dysfunctional, or uncapped, telomere ends, it has an inhibitory effect on the HR and A-NHEJ pathways and on the recombination of normal telomeres. Mouse embryonic fibroblasts (MEFs) deficient in Ku70 have normal telomere structure, however they exhibit increased sister telomere exchanges mediated by HR and chromosome fusions by the A-NHEJ pathway [156,157]. Additionally, Ku deficient S. cerevisiae display increased RAD52 dependent recombination of the subtelomeric DNA elements, consistent with its general HR inhibitory function [158,159]. While wildtype cells only exhibit long G-tails during late S phase, yeast Ku deficient strains possess long G-tails throughout the cell cycle. This defect is almost completely suppressed by the deletion of the 50 to 30 exonuclease Exo1, suggesting that Ku acts to inhibit inappropriate nucleolytic degradation of the C strand by nucleases [160]. Ku also positively regulates telomere length through telomere addition by aiding in the recruitment of telomerase. In yeast, Ku has been shown to interact with telomerase component 1 (TLC1), the telomerase RNA subunit, via binding of TLC1’s 48nt stem-loop region [161,162]. Deletion of the binding interface from either the Ku80 vWA domain or the TLC1 stem-loop results in decreased telomere length and decreased de novo telomere addition, perhaps due to the decreased levels of TLC1 RNA or altered localization of the telomerase holoenzyme [161,163–166]. There is no sequence conservation between TLC1 and the human telomerase RNA counterpart, hRT, however there is evidence that the interaction between the RNA component of human telomerase (hRT) and Ku in humans is conserved [167]. Another study detected an interaction with Ku and telomerase in human cells, however this interaction was reported to occur through the catalytic reverse transcriptase protein subunit hTERT [168]. The phenomenon of the telomere position silencing effect (TPE), observed in many eukaryotic species, is the process by which organisms transcriptionally silence genes located near the telomere (reviewed in [169,170]). In S. cerevisiae, Ku, along with the silent information regulator (Sir) complex, are essential for TPE, as deletion of either Ku subunit results in complete loss of telomeric silencing [171–174]. As a consequence of this role, Ku is also involved in the nuclear organization of telomeres. Telomeres and TPE proteins in S. cerevisiae are found distinctly clustered in foci around the nuclear periphery, and the loss of Ku function often results in the random distribution of telomeres throughout the nucleus [174–176]. Ku participates in these processes as part of multi-protein complexes with various proteins (such as the Sir proteins, Rap1 and Mps3) and Ku is particularly essential for their formation in the G1 phase of the cell cycle [176–178]. It is still currently unknown how Ku is associated with the telomere in vivo, whether it is through direct DNA binding of the end, or is mediated through a protein–protein interaction with another telomere bound factor. Ku has the ability to directly bind telomere DNA in vitro, in both mammals and yeast [179,180]. Furthermore, mutations in yeast Ku’s DNA binding domain reduces association with telomeric chromatin and renders it unable to protect the telomere end, suggesting that this region is key for telomere binding [181]. Despite this evidence, there is still some

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question whether it truly slides onto the telomere end via its DNA binding ring. Telomeres in many species form higher order loop structures designed to conceal the end from attack by Ku and the DSB repair pathways, so further research needs to be done to understand how and when Ku would be allowed access to the telomere end. Another possibility is that Ku is retained at the telomere through protein–protein interaction with other telomere bound factor(s). Ku has been shown to interact with TRF1, TRF2 and Rap1 [20,182–186]. These components of the shelterin complex bind DNA directly and then mediate the recruitment of other shelterin factors, such as TERF1-interacting nuclear factor 2 (TIN2) to the TRF proteins in humans and Sir3/4 to Rap1 in S. cerevisiae, and therefore could also be involved to recruit Ku at the telomeres [187,188]. A longstanding model for telomerase recruitment by Ku in yeast, suggested that Ku bound to the telomere end through its DNA binding ring and then bound the TLC1 RNA via the yeast Ku80 vWA domain [161]. However there is evidence that Ku requires the DNA binding domain for both DNA and RNA binding and cannot bind both simultaneously [189]. These observations suggest that Ku does not recruit telomerase to the telomere by binding the telomere directly, and instead favors a model where Ku must bind another telomere bound factor to be retained. A combination of DNA binding and protein interaction is also possible and has been reported for the shelterin component protection of telomeres 1 (POT1), which was shown to bind the G-strand of telomeres, as well as form multiple protein interactions [190,191]. Given that Ku has been shown to multimerize, it is possible that one Ku molecule interacts with the telomere end then multimerizes with another Ku molecule that is bound to telomerase. Although it appears that there are similarities in Ku telomere binding between yeast and mammalian systems, these organisms have different telomere structure and protein complexes present, so it is possible that Ku has different mechanisms for telomere binding and protection. Advances in high-resolution microscopy techniques have allowed for direct visualization of telomere structures, such as mammalian t-loop formation by TRF2, and could help in understanding Ku’s role at the telomere [192]. 6. Ku in disease 6.1. Immune system disorders The vertebrate immune system utilizes Ku and C-NHEJ to repair physiological DSBs generated to create immune system genetic diversity. Recombination of the V, D, and J segments of immunoglobulins in lymphoid cells and class switch recombination of the T-cell receptor genes in mature B cells allows for the recognition of a wide variety antigens, which results in an adaptive immune response [193]. Ku70 and Ku80 knockout mice display many immune system abnormalities including B cell developmental arrest, T cell arrest, and failed lymphocyte differentiation following g-irradiation [50,194–196]. Humans and mice suffering from genetic defects in V(D)J recombination present with a severe combined immunodeficiency (SCID) phenotype resulting in severe infections and poor prognosis. There are several characterized human disorders with various degree of SCID phenotype that have been linked to mutations in NHEJ factors, such as DNA Ligase IV, Artemis, and XLF, as well as the other DSB repair pathway and DDR factors [197–200]. Interestingly, only a few individuals have been reported to have genetic defects in the DNA-PK complex, however, all presented mutations occurred in the DNA-PKCS subunit, and thus far, no disorder linked to Ku mutations has been identified [201,202]. It is currently unknown whether mutations in Ku are better tolerated in humans, or in the contrary, whether deleterious Ku mutations are lethal in humans.

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6.2. Aging Aging is the progressive degeneration that occurs at the cellular and organismal level. One of the observed phenotypes of Ku deficient mice is increased aging and senescence [196,203–205]. Mice deficient in one Ku subunit have one-third the lifespan of their wildtype counterparts, partially in due to an early onset of the phenotypic symptoms of aging [206]. There is evidence that CNHEJ declines during the aging process, with lower efficiency observed in aging rodents, and in Alzheimer’s patients, so the loss of NHEJ in Ku deficient mice could be contributing to the aging process [207–209]. Furthermore, as previously described, Ku deficient mice and cells exhibit shortened telomeres as well as other telomere abnormalities. Shortened telomeres have been linked to increased aging and senescence, due to increased genomic instability leading to cell cycle arrest, so this could also be contributing to the aging phenotype observed in Ku-deficient mice [210,211]. 6.3. Cancer A common characteristic amongst human tumors is genomic instability [212]. Cells defective in DSB repair are predisposed to chromosome translocations and gene amplifications, potentially leading to the activation of oncogenes and tumorigenesis. Through its role in C-NHEJ, Ku functions as a cancer caretaker gene, promoting genomic integrity and preventing tumorigenesis. Ku knockout mice show increased chromosomal breakage, translocations and aneuploidy [196,203,205,206]. These mice display slightly increased cancer incidence as compared to controls, however this is mostly restricted to lymphomas [196,203,205,206]. Together with a p53 deficiency that impairs control of proliferation, Ku80 deficient mice show early onset tumor formation and further increased incidence of T and B cell lymphomas [51]. This suggests that Ku mutations can be oncogenic when accompanied by a mutation in a tumor suppressor gene. The expression of Ku has been frequently found deregulated in tumor samples and its expression level has been proposed as a marker of predicting patient response to radiation therapy and survival [213–218]. The exploitation of genetic defects in DNA repair and DDR factors have long been employed in cancer treatment [219]. As previously mentioned, Ku and DNA-PKCS deficient cell lines are sensitive to radiation and chemotherapeutic drugs, and therefore would make them potential druggable targets in cancer treatment. There are a few examples of strategies to target Ku specifically, including a subunit dimerization interference using a peptide, downregulation by RNAi and small molecule inhibition [220–223]. The therapeutic potential of targeting the Ku70/Bax interaction to activate apoptosis in cancer has been explored through creation of Bax inhibiting peptides derived from the Ku70 sequence and promoting Ku70 acetylation in vivo through deacetylase inhibitors [224–226]. A more commonly employed strategy to target the DNA-PK complex however, is to modulate the kinase activity of DNA-PKCS with small molecular inhibitors, as DNA-PKCS expression is frequently found upregulated in tumors [227]. 7. Conclusion Ku is a versatile, multifunctional protein that has integral roles in DNA repair, telomere maintenance and DNA damage signaling. The central importance of Ku to cellular homeostasis is best demonstrated by the knockout of Ku in mouse models, which results in a complex phenotype, highlighted by extreme immunodeficiency, susceptibility to cancer and accelerated aging. Interestingly, despite the identification of human diseases stemming

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from mutation in the majority of other important DSB and DDR factors, no Ku mutations have been observed in humans. This leads to the question as to whether Ku function is essential for human cell viability. Additionally, an unexplained phenotype observed in Ku deficient mice is that they are proportional dwarfs to their wildtype counterparts [196,205]. The cause of this phenomenon is currently unknown, but there are several studies identifying Ku as an interactor with proteins involved in gene transcription, DNA replication and cell cycle regulation (Table 1). These observations are currently not well understood but could point to even further roles for Ku in cellular maintenance. Overall, it is of critical importance to understand the differing roles of Ku at the cellular level and their implications in human disease.

[21]

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Acknowledgement

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This work was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to C.S.P.

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