REVIEW ARTICLE Repair of ionizing radiation-induced DNA double …€¦ · kinase) and WRN...

Biochem. J. (2009) 417, 639–650 (Printed in Great Britain) doi:10.1042/BJ20080413 639

REVIEW ARTICLERepair of ionizing radiation-induced DNA double-strand breaks bynon-homologous end-joiningBrandi L. MAHANEY*, Katheryn MEEK† and Susan P. LEES-MILLER†*1

*Department of Biochemistry and Molecular Biology and The Southern Alberta Cancer Research Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta,Canada T2N 4N1, and †College of Veterinary Medicine and Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, MI 48824, U.S.A.

DNA DSBs (double-strand breaks) are considered the mostcytotoxic type of DNA lesion. They can be introduced by externalsources such as IR (ionizing radiation), by chemotherapeuticdrugs such as topoisomerase poisons and by normal biologicalprocesses such as V(D)J recombination. If left unrepaired, DSBscan cause cell death. If misrepaired, DSBs may lead to chromo-somal translocations and genomic instability. One of the majorpathways for the repair of IR-induced DSBs in mammalian cells isNHEJ (non-homologous end-joining). The main proteins requiredfor NHEJ in mammalian cells are the Ku heterodimer (Ku70/80heterodimer), DNA-PKcs [the catalytic subunit of DNA-PK(DNA-dependent protein kinase)], Artemis, XRCC4 (X-ray-

complementing Chinese hamster gene 4), DNA ligase IV and XLF(XRCC4-like factor; also called Cernunnos). Additional proteins,including DNA polymerases μ and λ, PNK (polynucleotidekinase) and WRN (Werner’s Syndrome helicase), may alsoplay a role. In the present review, we will discuss our currentunderstanding of the mechanism of NHEJ in mammalian cellsand discuss the roles of DNA-PKcs and DNA-PK-mediatedphosphorylation in NHEJ.

Key words: DNA double-strand-break repair, DNA-dependentprotein kinase (DNA-PK), ionizing radiation, non-homologousend-joining, phosphorylation.

INTRODUCTION

DNA DSBs (double-strand breaks) are considered the most lethalform of DNA damage. They can be introduced by exogenousagents such as IR (ionizing radiation), topoisomerase poisons,radiomimetic drugs (e.g. bleomycin and neocarzinostatin), andby cellular processes such as V(D)J recombination, classswitch recombination, stalled replication forks and reactions thatgenerate ROS (reactive oxygen species) [1,2]. In the presentreview, we will focus on the detection and repair of IR-inducedDSBs by the NHEJ (non-homologous end-joining) pathway.

All organisms are exposed to low doses of naturally occurringIR, and IR is widely used in medical procedures such as X-raysand radiation therapy for the treatment of cancer patients [3–5]. IRdamages DNA by direct deposition of energy and also indirectlyby ionization of water molecules to produce hydroxyl radicalsthat attack the DNA. IR induces multiple forms of DNA damage,including damage to the bases and cleavage of the DNA backboneto form DNA SSBs (single-strand breaks). These types of DNAdamage are detected and repaired by the BER (base-excisionrepair) and SSB repair pathways respectively [6,7]. DSBs areformed when two SSBs occur on opposite DNA strands approx.10–20 bp apart. Thus IR-induced DSBs usually contain over-hanging 3′ and 5′ ends. In addition, the DNA termini frequentlycontain 3′-phosphate or 3′-phosphoglycolate groups, which must

be removed prior to ligation [8] (Figure 1A). Moreover, the DNAsurrounding the DSB may contain additional forms of DNA dam-age, producing what are termed complex or clustered lesions [9].If not repaired, such lesions can result in cell death. If misrepaired,DSBs have the potential to result in chromosomal translocationsand genomic instability [1,10].

In mammalian cells there are two major pathways for therepair of IR-induced DSBs, namely NHEJ and homologousrecombination or HDR (homology-directed repair) [2,4]. HDRis widely regraded as an accurate form of repair, which requiresan undamaged sister chromatid to act as a DNA template andfunctions only after DNA replication [2,11]. In contrast, NHEJ isactive throughout the cell cycle [12] and is considered the majorpathway for the repair of IR-induced DSBs in human cells [11]. Inits simplest sense, NHEJ entails straightforward ligation of DNAends. However, since the DNA ends formed by IR are complexand frequently contain non-ligatable end groups and other types ofDNA damage, successful repair of DNA lesions by NHEJ mustrequire processing of the ends prior to ligation. This can lead toloss of nucleotides from either side of the break, making NHEJpotentially error prone. In addition to HDR and NHEJ, thereis also increasing evidence for the existence of alternative end-joining pathways that directly ligate DNA ends in the absenceof NHEJ [13–17]; however, whether these pathways function innormal cells or only when NHEJ is deficient is not clear.

Abbreviations used: APLF, aprataxin and polynucleotide kinase-like factor; ATM, ataxia-telangiectasia mutated; ATR, ATM-, Rad3-related; BER, base-excision repair; BRCT, BRCA1 (breast-cancer susceptibility gene 1) C-terminal; CK2, casein kinase 2; DNA-PK, DNA-dependent protein kinase; DNA-PKcs,the catalytic subunit of DNA-PK; DSB, double-strand break; dsDNA, double-stranded DNA; FAT domain, FRAP (FKBP12-rapamycin-associated protein),ATM, TRRAP (transactivation/transformation-domain-associated protein) domain; FATC, C-terminal of FAT; FHA, forkhead-associated; HDR, homology-directed repair; IR, ionizing radiation; Ku, Ku70/80 heterodimer; MRN, Mre11–Rad50–Nbs1; NHEJ, non-homologous end-joining; PIKK, phosphoinositide3-kinase-like family of protein kinases; PKB, protein kinase B; PNK, polynucleotide kinase; SAP domain, SAF-A/B, Acinus and PIAS domain; SCID, severecombined immunodeficiency; SQ/TQ motif, serine residues or threonine residues that are followed by glutamine residues; SSB, single-strand break; Tdp1,tyrosyl-DNA phosphodiesterase 1; TdT, terminal deoxyribonucleotidyltransferase; WRN, Werner’s Syndrome helicase; XRCC4, X-ray-complementingChinese hamster gene 4; X4–L4 complex, XRCC4–DNA ligase IV complex; XLF, XRCC4-like factor.

1 To whom correspondence should be addressed (email [email protected]).

c© The Authors Journal compilation c© 2009 Biochemical Society

640 B. L. Mahaney, K. Meek and S. P. Lees-Miller

Figure 1 A model for NHEJ

(A) IR induces multiple forms of DNA damage including DSBs that contain non-ligatable end groups such as 3′-phosphate and 3′-phosphoglycolate groups (indicated by �). (B) The Ku heterodimer(orange) binds the ends of the DSB, tethering the ends together. Recruitment of Ku to the DSB occurs independently of other known NHEJ or DSB-repair proteins, consistent with Ku acting as thecornerstone of the NHEJ pathway. (C) Ku translocates inwards, allowing recruitment of DNA-PKcs (blue) such that it binds the extreme termini of the break (D). Recruitment of DNA-PKcs to the DSBrequires Ku, but no other NHEJ or DSB-repair factors. Two DNA-PK molecules (DNA-PKcs bound to DNA-bound Ku) interact to tether the DSB together in what has been termed a ‘synaptic complex’.This triggers autophosphorylation (yellow circles) of DNA-PKcs in trans (E), inducing a conformational change that causes release of the DNA ends and/or release of phosphorylated DNA-PKcsfrom the complex. Whether DNA-PKcs is released prior to recruitment of the X4–L4 complex (green) and its associated factors (F), or whether it remains part of a multi-protein complex until repair iscompleted (M) is not known. Inhibition of the protein kinase activity of DNA-PKcs (E), prevents dissociation of DNA-PKcs (F), blocking access of NHEJ or other repair factors to the DSB, resultingin radiation sensitivity. (G) A portion of the total cellular DNA-PKcs interacts with the nuclease Artemis (red), but if or when Artemis is released from the DNA-PKcs complex (H) is not known.(I) PNK (pink) interacts with XRCC4 suggesting that it is recruited to the break with the X4–L4 complex (green) (J). XLF (yellow) and DNA polymerase μ and λ (purple) interact with both X4–L4and Ku, suggesting that they are recruited after or at the same time as X4–L4 is recruited to the Ku–DNA complex (K). Other processing enzymes such as WRN and APLF (shown in grey) may alsobe recruited through interactions with DNA-bound Ku, XRCC4 and/or the X4–L4 complex (L). The order of recruitment of processing factors may be flexible and depend on the precise type of DNAdamage present at the DSB. Multiple protein–protein and protein–DNA interactions may stabilize the formation of the complex at the DSB as well as aid in retention of NHEJ factors at the break. Oncethe ends are processed, the X4–L4 complex ligates the ends, repairing the break. Ligation of incompatible DNA ends is aided by the regulatory factor, XLF (K). How the various factors are releasedafter repair is unknown, however, it is possible that ubiquitylation (Ub) and/or proteolysis may be involved (N). Reactions requiring or enhanced by the presence of DNA-bound Ku are shown in red.An animated version of this Figure is available at http://www.BiochemJ.org/bj/bj417/0639/bj4170639add.htm.

THE NHEJ PATHWAY IN MAMMALIAN CELLS

NHEJ in mammalian cells requires Ku (Ku70/80 heterodimer),DNA-PKcs [the catalytic subunit of DNA-PK (DNA-dependentprotein kinase)], XRCC4 (X-ray-complementing Chinese hamstergene 4), DNA ligase IV, Artemis and XLF (XRCC4-like factor;also called Cernunnos). Deletion or inactivation of any of thesecore NHEJ factors induces marked sensitivity to IR and otherDSB-inducing agents, as well as defects in V(D)J recombination[18–21]. Increasing evidence suggests that additional DNA-processing enzymes, such as DNA polymerases μ and λ, PNK(polynucleotide kinase) and WRN (Werner’s syndrome helicase)also play a role in NHEJ, at least at a subset of DNA ends. Ingeneral terms, NHEJ is thought to proceed through the followingstages: (i) detection of the DSB and tethering/protection of the

DNA ends; (ii) DNA end-processing to remove damaged or non-ligatable groups; and (iii) DNA ligation. In the following sections,we will review the roles of the main players in NHEJ in each ofthese steps and propose a model for how they may function inNHEJ. Since the protein kinase activity of DNA-PKcs is requiredfor NHEJ [22,23], we will also discuss the role of DNA-PK-mediated phosphorylation in the process.

Detection of the DSB and tethering of the DNA ends

The Ku70/80 heterodimer

The first step in NHEJ is detection of the DSB by Ku (Figure 1B).Ku is composed of Ku70 and Ku80 subunits, each of which


Repair of IR-induced DSBs by NHEJ 641

Figure 2 Major features of Ku70 and Ku80 polypeptides

Domain boundaries, phosphorylation sites (red), protein–protein interaction sites and interactingproteins (yellow ovals) are shown for (A) Ku70 and (B) Ku80. Domain boundaries forthe von Willebrand domain (vWa) (amino acids 35–249), Ku core (amino acids 266–529)and SAP domains (amino acids 573–607) of Ku70 and the vWa (amino acids 7–237), Kucore (amino acids 244–543) and C-terminal domains (amino acids 590–709) of Ku80 wereobtained from the NCBI database. The location of putative nuclear localization sequences(NLS) in Ku70 (amino acids 539–556) and Ku80 (amino acids 561–569) are from [180,181].In vitro DNA-PK phosphorylation sites in Ku70 (Ser6) and Ku80 (Ser577, Ser580 and Thr715)are indicated in red [63]. Amino acids 720–732 of Ku80 contain the DNA-PKcs binding region[29,30].

contributes to a central DNA-binding core [24]. In addition, theN- and C-terminal regions of Ku70 and Ku80 contain uniqueregions. The N-terminus of Ku70 contains an acidic domain thatis phosphorylated in vitro by DNA-PKcs [25], whereas the C-ter-minus contains an SAP (SAF-A/B, Acinus and PIAS) domainwhich is a putative chromatin/DNA-binding domain (reviewed in[26]) (Figure 2A). The C-terminal region of Ku80 forms a longflexible arm that may be involved in protein–protein interactions[27,28] and, at the extreme C-terminus, a conserved region whichis required for interaction with DNA-PKcs [29–31] (Figure 2B).In vitro, Ku binds to ends of dsDNA (double-stranded DNA) withhigh affinity and without apparent sequence specificity (reviewedin [32]). This property is due to the structure of the Ku70/80 DNA-binding core, which adopts a pre-formed loop that encircles theDNA [24] (Figure 1B). Binding of Ku to the DSB ends may assistin tethering the broken ends together [33]. Once bound, Ku trans-locates inwards from the DNA end (Figure 1C) making theextreme termini accessible to other proteins, such as DNA-PKcs[34] (Figure 1D, and described in detail below).

Recent studies using laser microbeam irradiation to induceDNA damage in the nuclei of living cells have shed light on theorder of recruitment of NHEJ factors to sites of DNA damageas well as the kinetics of the repair process. Consistent withKu being the major DSB-sensing protein in NHEJ, fluorescentlytagged Ku localizes to sites of laser-induced DNA damage incells within a few seconds and independently of other NHEJ- orDSB-repair proteins [35,36]. Recruitment of Ku to the DSB alsoserves to recruit other NHEJ proteins to the DSB. As discussedbelow, Ku interacts with DNA-PKcs (reviewed in [26,37]), theX4–L4 (XRCC4–DNA ligase IV) complex [36,38,39], XLF [40],DNA polymerase μ [41] and DNA polymerase λ [42] in vitro.The interactions of DNA-PKcs [43], XLF [44], DNA polymeraseμ [42] and DNA ligase IV [38] with Ku are facilitated by, orenhanced in the presence of, DNA, suggesting that binding of Kuto DNA is a prerequisite for interaction with other NHEJ proteins.Interestingly, binding of Ku to DNA results in a conformationalchange in the flexible C-terminal regions of both Ku70 and Ku80,which might facilitate its interactions with partner proteins [45].Ku is also required for the recruitment of DNA-PKcs [46], XRCC4[36] and XLF [40] to sites of DNA damage in vivo. Thus Ku canbe regarded as the cornerstone of NHEJ.

Figure 3 Major features of DNA-PKcs

Domain boundaries and major features are represented as in Figure 2. The N-terminal domainextends from amino acids 1–2908, the FAT domain is from amino acids 2908–3539, the PIKKdomain from amino acids 3645–4029 and the FATC domain from amino acids 4906–4128.In vivo phosphorylation sites between Thr2609 and Thr2647 (termed the ABCDE cluster) are from[21,68]. In vivo phosphorylation sites between Ser2023 and Ser2056 (the PQR cluster) arefrom [81]. The 2671 cluster, which contains four sites between Thr2671 and Thr2677 is from [73].In vivo phosphorylation of Thr3950 and Ser3205 have been described in [80] and [57] respectively.Reported interaction sites for Ku are from [50] (amino acids 3002–3850) and the putative PIKKregulatory domain (PRD) is from [51].

DNA-PKcs

One of the first proteins shown to interact with Ku was DNA-PKcs [43]. DNA-PKcs, the product of the PRKDC gene, isa large polypeptide of over 4000 amino acids, and a memberof the PIKKs (phosphoinositide 3-kinase-like family of proteinkinases) (reviewed in [26,47]). The N-terminal ∼250 kDa ofDNA-PKcs contains a putative DNA-binding domain [48], aleucine-rich region and a series of HEAT [huntingtin, elongationfactor 3, A subunit of protein phosphatase 2A and TOR1 (targetof rapamycin 1)] repeats [49], but few other distinguishingfeatures (Figure 3). The C-terminal region contains a FAT[FRAP (FKBP12-rapamycin-associated protein), ATM (ataxia-telangiectasia mutated), TRRAP (transactivation/transformation-domain-associated protein)] domain that is characterized by weakamino acid similarity to other members of the PIKK family,followed by a kinase domain and a C-terminal FATC domain(Figure 3). Cells that lack DNA-PKcs are highly radiosensitiveand have defects in V(D)J recombination, specifically inprocessing of coding joints. Moreover, in mice, dogs and horses,DNA-PKcs deficiency is associated with SCID (severe combinedimmunodeficiency) (reviewed in [20,21]).

The interaction between DNA-PKcs and Ku is mediated bya conserved region in the extreme C-terminus of Ku80 [29–31](Figure 2B), and C-terminal regions of DNA-PKcs have beenimplicated in its interactions with Ku [50,51] (Figure 3). Kuand DNA-PKcs only interact in the presence of DNA [52], andrecruitment of DNA-PKcs to sites of DNA damage in vivo is Ku-dependent [46]. Inward translocation of Ku allows DNA-PKcs tointeract with the extreme termini of the DNA [34], allowing twoDNA-PKcs molecules to interact across the DSB in a so-called‘synaptic complex’ [53] (Figure 1D). This interaction stimulatesthe kinase activity of DNA-PKcs [53], promoting phosphorylationin trans across the DSB [54] (discussed in more detail below).Once assembled at the DNA ends, the DNA-PKcs–Ku–DSBcomplex serves to tether the ends of the DSB together and isthought to protect the DNA ends from nuclease attack.

The protein kinase activity of DNA-PKcs

DNA-PKcs has weak serine/threonine kinase activity that isgreatly enhanced in the presence of dsDNA ends and Ku [43].The DNA-PKcs–Ku–DNA complex is referred to as DNA-PK



(Figure 1D). Like other members of the PIKK family, DNA-PK phosphorylates many of its substrates on serine residues orthreonine residues that are followed by glutamine residues (SQ/TQ motifs) [55,56]; however, DNA-PK also phosphorylatesproteins on non-SQ/TQ sites in vitro [25,57–59]. Significantly,the protein kinase activity of DNA-PKcs is required for NHEJ[22,23], therefore identification of its physiological targets iscritical to understanding its function in NHEJ. Moreover, inhib-itors of DNA-PK kinase activity radiosensitize cells and inhibitDSB repair, making DNA-PK a possible therapeutic target[60,61].

Given its role in NHEJ, obvious candidates for physiologicalsubstrates of DNA-PKcs are other NHEJ factors. However,although DNA-PK phosphorylates Ku70, Ku80, XRCC4, XLF,Artemis and DNA ligase IV in vitro, there is little evidencethat any of these phosphorylation events are required forNHEJ in vivo [58,59,62–64]. To date, the best candidatesubstrate for DNA-PK is DNA-PKcs itself. Sixteen in vitroautophosphorylation sites in DNA-PKcs have been identified[57,65–67], and it is likely that additional sites exist [20].DNA-PKcs is also phosphorylated in response to DNA damagein vivo. Studies from several laboratories including our ownhave shown that phosphorylation of Ser2612 and Ser2624 andThr2609, Thr2620, Thr2638 and Thr2647 (which we have termed theABCDE cluster [68]), as well as Ser2056 and Thr3950, are all IR-inducible and DNA-PK-dependent in vivo [54,66,67,69] (Fig-ure 3). Similarly, a proteomics study has reported that IR-inducedphosphorylation of Ser2612, and Thr2638 and Thr2647 occurs in cellsin which the activity of the related protein kinase ATM is inhibited,again consistent with DNA-PK-dependent phosphorylation atthese sites in vivo [70]. However, other studies have reportedthat ATM and the related PIKK, ATR (ATM-, Rad3-related), canphosphorylate Ser2612, and Thr2609, Thr2638 and Thr2647 in responseto IR or UV respectively [71,72]. It is possible that all three PIKKscontribute to the phosphorylation of DNA-PKcs in vivo, depend-ing on cell type, stage of cell cycle, and/or the type or extent ofDNA damage. Additional in vivo phosphorylation sites on Ser2671,Ser2674, Ser2675, Ser2677 [73] and Ser3205 [74] have been identified inproteomics screens (Figure 3); however, the kinases responsibleand effects of phosphorylation at these sites on function is notknown.

In vitro, autophosphorylation of DNA-PKcs results in loss ofprotein kinase activity and dissociation of phosphorylated DNA-PKcs from DNA-bound Ku (Figures 1E and 1F), suggesting thatautophosphorylation of DNA-PKcs may serve to regulate thedisassembly of the DNA-PK complex [75,76]. Significantly, cellsexpressing DNA-PKcs in which serine residues and threonineresidues in the ABCDE cluster have been mutated to alanine aremore radiosensitive than cells expressing no DNA-PKcs at all[68]. The rate of the alternative DSB-repair pathway, HDR, is alsoreduced in these cells [77]. Similarly, cells in which the proteinkinase activity of DNA-PK is inhibited by a small moleculeinhibitor are more radiosensitive than DNA-PKcs-null cellsand have reduced rates of HDR [78]. Moreover, althoughpurified DNA-PKcs containing alanine mutations at the ABCDEphosphorylation sites is kinase active, it has reduced ability todissociate from DNA-bound Ku in vitro [79,80]. Taken together,these results support a model in which autophosphorylationof DNA-PKcs is required for its release from DSBs in vivo (Fig-ures 1E and 1F). Recent studies using laser microbeam irradiationto induce DNA damage in cells expressing fluorescently taggedDNA-PKcs also support this model, in that autophosphorylation-defective DNA-PKcs, as well as kinase-dead DNA-PKcs, wereretained significantly longer at sites of laser-induced DNA damagethan wild-type DNA-PKcs [46]. It should also be noted however,

that the effects of autophosphorylation on DNA-PKcs functionmay be highly complex in vivo, since phosphorylation at otherregions of the molecule enhance the rate of HDR and phos-phorylation at different sites can either positively or negativelyaffect DNA end-processing [81] (discussed in detail in[20]).

Processing of DNA ends

Once the DNA ends have been detected and secured, the nextstep in NHEJ is thought to be processing of the DNA termini toremove non-ligatable end groups and other lesions. IR-inducedbreaks are complex and may be highly variable from one DSB toanother. Depending on the nature of the break, different processingenzymes may be required to remove blocking end groups, fillin gaps, and/or remove damaged DNA or secondary structureelements surrounding the break. Therefore repair of differentbreaks may require different combinations of processing enzymes.Since NHEJ occurs in the absence of a DNA template or extendedregions of microhomology, processing of the DNA ends hasthe potential to result in loss of nucleotides, making NHEJ aninherently error-prone process. Indeed, both in vitro assays andcell-based assays show that NHEJ proceeds with loss of sequencefrom DNA ends, and that this process is regulated, at least in part,by DNA-PKcs [68,81,82]. Candidate processing enzymes includeArtemis, DNA polymerases μ and λ, PNK, and possibly APLF(aprataxin and PNK-like factor) and WRN.

Artemis

Artemis possesses 5′→3′ exonuclease activity and, in thepresence of DNA-PKcs and ATP, acquires endonuclease activitytowards DNA-containing dsDNA/ssDNA (single-stranded DNA)transitions as well as DNA hairpins [83,84]. Artemis can alsoremove 3′-phosphoglycolate groups from DNA ends in vitro,again consistent with a role in SSB or DSB repair [85]. Artemisis composed of an N-terminal metallo β-lactamase/β-CASPnuclease domain [86] and a C-terminal region of uncertainfunction that is highly phosphorylated both in vitro and in vivo(discussed below) (Figure 4A). Inactivation of Artemis resultsin RS-SCID (radiation-sensitive SCID) in humans, and, similarto cells lacking DNA-PKcs, cells lacking Artemis accumulateunopened DNA hairpins at unprocessed coding joints duringV(D)J recombination [87,88]. Moreover, Artemis interacts withDNA-PKcs providing a mechanism whereby it may be recruitedto the DSB [83,89] (Figure 1G). However, although Artemis-deficient cells are radiation-sensitive they do not have majordefect in DSB repair, suggesting that Artemis is required forthe repair of only a subset of DNA-damage events in vivo[90,91].

Both DNA-PKcs and ATM phosphorylate Artemis in vitro[62,65], and Artemis is highly phosphorylated at both basal andDNA-damage-induced sites in vivo [62,89–94] (Figure 4A). Ithas been suggested that phosphorylation of Artemis by DNA-PKcs is required for the endonuclease activity of Artemis [83,95];however, mutation of DNA-PKcs/ATM phosphorylation sites inArtemis had no effect on its endonuclease activity in vitro [62].Instead, we and others have proposed that autophosphorylationof DNA-PKcs may be required for the endonuclease activity ofArtemis, by facilitating access of Artemis to its DNA substrates[62,96].

The role of phosphorylation on Artemis function is far fromclear. It has been suggested that DNA-PK kinase activity is re-quired for recruitment of Artemis to damaged DNA [97], whereasother studies suggest that DNA-damage-induced phosphorylation



Figure 4 Major features of the processing enzymes Artemis, polymeraseX family members μ and λ, PNK and APLF

Domain boundaries and major features of Artemis, PNK, APLF, polymerase μ and polymerase λ

are represented as in Figure 2. (A) Artemis: the metalloβ-lactamase domain (amino acids 1–155)and β-CASP domain (amino acids 156–385) are as described in [86]. Amino acids 398–403are required for interaction with DNA-PKcs [89,95]. The C-terminal region of Artemis is highlyphosphorylated at multiple sites both in vitro and in vivo [62,65,89,90,92,93], but the effectsof phosphorylation on function are not known. (B and C) Polymeraseμ andλ: domain boundariesfor the lyase (amino acids 156–227) and polymerase (amino acids 227–494) domains ofpolymerase μ are based on a structure-based alignment of polymerase X family members[103,182]. The BRCT (amino acids 29–109) domain is as described in the NCBI database.The lyase (amino acids 242–327), polymerase (amino acids 327–575) and the BRCT domains(amino acids 35–125) of polymerase λ are from [103,182]. (D) PNK: domain boundariesfor the FHA (amino acids 6–110), phosphatase (amino acids 145–337) and kinase (aminoacids 341–521) domains are based on the X-ray crystal structure [183]. CK2-phosphorylatedXRCC4 and XRCC1 interact with the FHA domain [109,174]. Ser114 and Ser126 are IR-inducedphosphorylated sites of unknown function [70]. (E) APLF: amino acids 20–102 compose theFHA domain. XRCC1/XRCC4 bind within the FHA domain [113,114]. The poly(ADP-ribose)binding zinc finger (PBZ) regions (amino acids 377–398 and amino acids 419–440) of APLFare shown in purple [115]. Although APLF has endonuclease and exonuclease activity, thesedomains have yet to be defined.

of Artemis in vivo is largely ATM-dependent ([90–93,98] andR. Ye, S. Hiebert and S. P. Lees-Miller, unpublished work).However, it is possible that DNA-PKcs may phosphorylateArtemis at high doses of IR and/or in ATM-deficient cells ([91,94],and R. Ye, S. Hiebert and S. P. Lees-Miller, unpublished work).Moreover, Artemis has also been shown to function in an ATM-dependent pathway for the repair of a subset of complex DNAlesions in vivo [100]. Thus it seems likely that Artemis is involvedin multiple aspects of the DNA-damage response and that it’sactivity may be regulated by ATM and/or DNA-PK.

DNA polymerases μ and λ

Processing of complex, IR-induced DNA damage can lead tothe creation of DNA gaps that require the action of DNA poly-merases for their repair. Members of the DNA polymeraseX family of DNA polymerases, polymerase μ, polymerase λand TdT (terminal deoxyribonucleotidyltransferase), have allbeen implicated in NHEJ. TdT interacts with Ku, but is onlyexpressed in lymphocytes and so its function is limited to V(D)Jrecombination (reviewed in [101]). In contrast, polymerase μand polymerase λ are widely expressed and are thought to havemore widespread roles in NHEJ (reviewed in [101]). DNA poly-merases μ and DNA polymerase λ each contain an N-terminalBRCT [BRCA1 (breast-cancer susceptibility gene 1) C-terminal]domain that is required for their functions in NHEJ (Figures 4Band 4C). DNA polymerases μ and λ are recruited to DSBs viatheir interactions with Ku and the X4–L4 complex (reviewed in[102]) (Figure 1K). Which polymerase is recruited to the DSBmay depend on the type of damage to be repaired. Although poly-merase μ and polymerase λ carry out similar gap-filling reactions,they differ in their requirement for a DNA template. Polymerase λis largely template-dependent [103,104], whereas polymeraseμ is less template-dependent [104,105] and has the unique abilityto direct template-independent synthesis across a DSB with noterminal microhomology [106]. Precisely when polymerase μ andpolymerase λ are recruited to the DSB and whether their recruit-ment requires Ku or X4–L4 in vivo remains to be determined.Since cells lacking one or both polymerases are not highly sensit-ive to IR [107], it seems likely that polymerase μ and polymeraseλ are only required for the repair of a small subset of DNA breaks(reviewed in [105]). DNA polymerase λ is phosphorylated inresponse to IR [70], but the kinase(s) responsible and the effectson function are not known.

PNK

PNK has both 3′-DNA phosphatase and 5′-DNA kinase activities,and thus is ideally suited to remove non-ligatable end groupsfrom DNA termini (reviewed in [108]). Indeed, several studieshave pointed to a role for PNK in NHEJ. First, the N-terminalFHA (forkhead-associated) domain of PNK (Figure 4D) interactswith CK2 (casein kinase 2)-phosphorylated XRCC4, providing apotential mechanism to recruit PNK to the DSB [109] (Figures 1Iand 1J). In vitro DSB end-joining studies also indicate a role forPNK in NHEJ [110]. Moreover, knockdown of PNK in humancells renders them radiosensitive and defective in DSB repair[111]. Radiation sensitivity was attributed to a defect in NHEJ,since PNK-knockdown cells were proficient at sister chromatidexchange, but epistatic with the DNA-PKcs-defective cell line,M059J [112]. PNK is also phosphorylated in vivo in responseto IR [70]; however, the kinase(s) responsible and the effects onfunction are not known.

APLF

A potential new player in NHEJ is APLF [113–115] (also calledC2orf13, Xip1 [116] and PALF [117]). APLF has both endo-nuclease and exonuclease activities, consistent with a role inprocessing DNA ends [117]. APLF contains a PNK-like FHAdomain, and, like PNK, interacts with CK2-phosphorylatedXRCC4 [113,114,116,117] (Figure 4E). APLF also interacts withKu, and down-regulation of APLF causes defects in DSB repair[113,114,117]. Taken together, these results support a possiblerole for APLF in NHEJ (Figure 1L). APLF is phosphorylated inan ATM-dependent manner in response to DNA damage, but thefunction of phosphorylation is not known [114,116].



WRN

WRN is a member of the RecQ helicase family that possessesDNA-dependent ATPase, 3′→5′ DNA helicase, strand annealingand 3′→5′ exonuclease activities. Inactivation of WRN is asso-ciated with premature aging, cancer predisposition and genomicinstability (reviewed in [118]). WRN interacts with Ku whichstimulates its exonuclease activity [119–121]. WRN is phos-phorylated in vitro by DNA-PK and is phosphorylated in a DNA-PK-dependent manner in cells [122]. WRN also interacts with theX4–L4 complex which also stimulates its exonuclease activityin vitro [123]. Thus, although WRN-negative cells are not highlyradiation-sensitive [118], several lines of evidence support a rolein NHEJ (Figure 1L).

Other potential processing enzymes

It is possible that other processing enzymes may also play a role inNHEJ. One candidate, Tdp1 (tyrosyl-DNA phosphodiesterase 1)removes 3′-phosphoglycolate groups from DNA ends [124];however, recent studies suggest that Tdp1 is primarily involved inthe repair of SSBs, not DSBs [125]. Another possible processingenzyme is the Mre11 nuclease; however, although Mre11 isrequired for NHEJ in Saccharomyces cerevisiae [126], it is notthought to play a role in vertebrate NHEJ [127].

Logistically, end-processing must occur prior to ligation of theDNA ends; however, precisely when the processing enzymes arerecruited to the DSB is not clear. It is possible that many aspectsof DNA end-processing occur within a multi-protein complexcomposed of Ku, XRCC4, DNA ligase IV and possibly DNA-PKcs, that is assembled at the DSB (Figure 1). It is also possiblethat the specific enzymes involved in end-processing and theirorder of recruitment to the DSB may be quite flexible, dependingon the nature of the break and other factors [128,129].

Ligation of the DNA ends

The X4–L4 complex

Once the DNA ends have been processed they must be ligatedto repair the DNA. Ligation is carried out by DNA ligase IV,which exists in complex with XRCC4 (referred to here as X4–L4) (Figure 1J).

XRCC4 is required for both NHEJ and V(D)J recombination[130,131]. It is composed of a globular head domain, an elongatedα-helical stalk and a C-terminal region of unknown function [132](Figure 5A). XRCC4 has no known enzymatic activity, but ratheracts as a scaffolding protein, facilitating the recruitment of otherNHEJ proteins to the break. XRCC4 itself is a homodimer and twodimers can interact to form tetramers [132,133]. The most well-characterized binding partner of XRCC4 is DNA ligase IV. DNAligase IV contains two C-terminal BRCT domains separated by alinker region that interacts with the α-helical region of XRCC4 toform a highly stable complex [134,135] (Figure 5B). XRCC4stabilizes DNA ligase IV and stimulates its activity [136,137].Interestingly, DNA ligase IV has the unusual property of beingable to ligate one DNA strand at a time [138], perhaps allowingprocessing enzymes to act simultaneously on end groups on theopposite strand.

Consistent with its role as a scaffolding protein, XRCC4 and/orthe X4–L4 complex interacts with Ku [36,38,39,139], PNK [109],APLF [113,114] and XLF [140–142], as well as with DNA [143];however, precisely when the X4–L4 complex (and presumably itsassociated factors) is recruited to the DSB is not clear. NHEJ hasbeen assumed to proceed in a stepwise fashion with binding of Kuand DNA-PKcs, followed by recruitment of the X4–L4 complex

Figure 5 Major features of XRCC4, DNA ligase IV and XLF

Domain boundaries and major features of DNA ligase IV, XRCC4 and XLF are representedas in Figure 2. (A) XRCC4: the head (amino acids 1–115) and stalk domains (amino acids135–233) of XRCC4 are based on the X-ray crystal structure [184]. The region of XRCC4required for dimerization (amino acids 119–155) is from [132]; DNA ligase IV interacts withXRCC4 between amino acids 173–195 [134]; CK-2 phosphorylates XRCC4 at Ser233 [109]and DNA-PK phosphorylates XRCC4 at Ser260 and Ser318 in vitro [58]. The XLF-binding site(amino acids 63–99) is from [150]. (B) DNA ligase IV: the N-terminal (amino acids 14–203) andligase domain (amino acids 248–451) boundaries are as found in the NCBI database. TheXRCC4-binding site in DNA ligase IV (amino acids 755–782; [134]) is located betweenthe BRCT domains (amino acids 661–731 and 829–898); in vitro, DNA ligase IV isphosphorylated at Thr650, Ser668 and Ser672 [64]. (C) XLF: the head (amino acids 1–135)and stalk (amino acids 135–233) domains are based on the X-ray crystal structure [184]. Leu115

is crucial for XRCC4 binding and amino acids 125–224 are involved in homodimerization[150,184]. In vivo, XLF is phosphorylated at Ser245 by DNA-PK and Ser251 by ATM but the effectof phosphorylation on function is unknown [59].

[26,37]. Indeed, biochemical studies suggest that DNA-PKcs isrequired for recruitment of the X4–L4 complex to chromatinafter damage [144]; however, recent laser microbeam irradiationexperiments have suggested that, although recruitment of XRCC4to sites of damage requires Ku, it does not require DNA-PKcs[36,44] (although localization at the break may be stabilized bythe presence DNA-PKcs [44]). Thus it is possible that DNA-PKcsand the X4–L4 complex may be recruited to the DSB indepen-dently, rather than in a sequential manner, as was originallysupposed (Figure 1).

XRCC4 is highly phosphorylated in vivo [130] and its phos-phorylation, as detected by a mobility shift on SDS/PAGE, isenhanced by DNA damage [144,145]. It has been suggested thatDNA-PK is required for DNA-damage-induced phosphorylationof XRCC4 [144,145] and that DNA-PKcs promotes ligation byX4–L4 [146]. Indeed, XRCC4 is phosphorylated by DNA-PKin vitro [130,133] (Figure 5A). However, DNA-PK phosphoryl-ation sites in XRCC4 are not required for either NHEJ or V(D)Jrecombination [58,147], and the role for DNA-PK-mediatedphosphorylation in the function of XRCC4 still remains unclear.As discussed above, XRCC4 is also phosphorylated by CK2 [109],which creates a binding site for the FHA domain of PNK[109] and APLF [113,114,116,117], facilitating their recruitmentto the DSB. XRCC4 is also SUMOylated in vivo, and thismodification is important for nuclear localization of XRCC4 andDSB repair [148].



XLF

XLF is similar in structure to XRCC4 [140,149,150] (Figure 5C),interacts with XRCC4 in vitro [140,149] and is required for NHEJand V(D)J recombination [140–142]. In vitro, XLF stimulates theactivity of DNA ligase IV towards non-compatible DNA ends,suggesting that XLF may only regulate the activity of X4–L4under a subset of conditions [138,149,151–154]. Like XRCC4,XLF also interacts with DNA. This interaction is highly dependenton the length of the DNA molecule and is enhanced by Ku[40,151]. Surprisingly, given the ability of XLF to interact withXRCC4, XRCC4 was not required for the recruitment of XLFto sites of DNA damage in vivo [40]. However, the presence ofXRCC4 did result in XLF being retained longer at the damagesites, suggesting that XLF is recruited to the DSB through inter-action with DNA-bound Ku, but stabilized at the break byinteraction with X4–L4. Like XRCC4, XLF is phosphorylatedin vitro at C-terminal sites by DNA-PK (Figure 5C) and is phos-phorylated by both ATM and DNA-PK in vivo; however,phosphorylation is not required for NHEJ and its function remainsunclear [59].

CONCLUSIONS

In conclusion, NHEJ has emerged as one of the major pathwaysfor the repair of IR-induced DSBs in mammalian cells. Many ofthe proteins required for NHEJ have been identified and charac-terized at a biochemical and/or cellular level and, in many cases,animal models have been generated (reviewed in [19]). However,although many protein–protein and protein–DNA interactionshave been identified at the biochemical level, until recently howthe NHEJ proteins interact in the cell has been largely unknown.The emerging challenge is to understand the choreography anddynamics of recruitment and release of each of the NHEJ factorsto the DSB in vivo.

As discussed above, the use of laser microbeam irradiation toinduce DNA damage in living cells has provided intriguing newinsights into the interplay between the various components of theNHEJ reaction. However, it should be noted that laser microbeamirradiation can introduce large, perhaps non-physiological,amounts of damage within the nucleus (discussed in [35,46,155]).Indeed, some studies have reported that DNA-PKcs and Ku onlylocalize to laser-induced sites of DNA damage when high-powerlasers are used [156]. Other approaches, such as the introductionof defined DSBs in the genome followed by ChIP (chromatinimmunoprecipitation) analysis are also beginning to yield newinformation on the kinetics of DSB repair in the cell and theproximity of various proteins to the break [157,158], and arelikely to be important tools as researchers tease apart the kineticsof the various DSB-repair pathways. However, these methods alsohave drawbacks. For example, DSBs created by endonucleases arenot formed instantaneously and the DNA ends at these breaksare complementary and the bases unmodified, unlike IR-inducedDSBs, which are often not directly ligatable, complex and createdmuch more rapidly. Furthermore, in yeast, IR-induced and endo-nuclease-induced DSBs are differentially processed in a cell-cycle-dependent manner [159].

The rapid pace of discovery in this field means that new modelsfor NHEJ continue to be developed, and that new questionsare raised. As discussed above, recent studies confirm that Kuplays a central role not only in detection of the DSB but alsoin the recruitment and or stabilization of other NHEJ proteins atthe break. However, some experiments have questioned whetherrecruitment of DNA-PKcs is required for recruitment of the X4–L4 complex to the DSB, and it is unclear whether DNA-PKcs

is required only for initiation of end-processing (Figure 1F), orwhether the holoenzyme remains assembled at the DSB untilrepair is complete (Figure 1M). In addition, a picture has emergedin which end-processing (trimming, fill-in and ligation of eachseparate strand) may occur in a flexible manner. Thus differentproteins may be recruited depending on the nature of the break.One intriguing and still unanswered question in NHEJ is how Kuis released from the DNA prior to ligation. The structure of thecore DNA-binding domain of Ku70/80 suggests that either Kumust back off the DNA prior to ligation or that it is removed fromthe DNA by proteolysis. A recent study has shown that Ku80 ismodified by ubiquitylation in vitro, and that this has the potentialto regulate the release of Ku from DNA, at least in cell extracts[160] (Figure 1N).

Although it is clear that cells which lack DNA-PKcs or in whichDNA-PK activity has been inhibited are highly radiosensitive andhave defects in DSB repair, the precise role of DNA-PKcs in NHEJis still not fully understood. Studies from several laboratories,including our own, have shown that the major role of DNA-PKcsin NHEJ appears to be autophosphorylation-dependent regulationof access to the DNA ends [20,21,62]. Therefore DNA-PKcsprobably plays a regulatory role in NHEJ. This is consistent withthe fact that, unlike Ku, XRCC4 and DNA ligase IV, DNA-PKcs isnot conserved in evolution and seems only to be required for NHEJin higher eukaryotes (discussed in [161]). In the absence of a high-resolution structure for DNA-PKcs or the DNA-PKcs–Ku–DNAcomplex, precisely how DNA-PKcs interacts with Ku and DNAis still unclear. Low-resolution structures suggest that DNA-PKcsbinds dsDNA via a central cavity, consistent with a role forDNA-PKcs in the protection of DNA ends [162]. Moreover,a low-resolution structure of the DNA-PKcs–Ku–DNA com-plex is consistent with interaction of DNA-PKcs and Ku withDNA ends to form a synaptic complex as suggested in Figure 1(E)[163,164]. We speculate that, after assembly of the complex,autophosphorylation of DNA-PKcs induces a conformationalchange that releases DNA-PKcs from the Ku–DNA complex,thus making the DNA ends accessible for downstream processingenzymes.

It has also become clear that DNA-PKcs does not have amajor role in phosphorylating other components of the NHEJpathway and/or that phosphorylation of NHEJ factors by DNA-PK is not required for NHEJ. This leads to the question ofwhether the main substrate of DNA-PKcs is itself, or whether,like ATM, it does indeed phosphorylate multiple substrates in vivo[165–167]. If the latter, then it seems likely that additionalphysiological substrates of DNA-PK might be found outside thecanonical NHEJ pathway. Indeed, like ATM and ATR, DNA-PK contributes to the DNA-damage-induced phosphorylationof histone H2AX on Ser139 [168]. This phosphorylated form ofH2AX, termed γ -H2AX, is widely regarded as a marker forunrepaired DSBs, and clusters of γ -H2AX molecules, termedfoci, serve to recruit and/or retain other DSB-repair proteins at thesites of DNA damage [169]. Another recently identified substrateof DNA-PKcs is the pro-survival protein kinase PKB (proteinkinase B)/Akt. DNA-PKcs was shown to interact with PKB/Aktand was required for DNA-damage-induced phosphorylation andactivation of PKB/Akt [170]. It is possible that proteomicapproaches may yield additional DNA-PK substrates. A recentproteomics screen in which antibodies to phosphorylated SQ/TQpeptides were used to immunoprecipitate proteins from irradiatedcells identified over 900 IR-induced phosphorylation sites in over600 protein substrates [70]. Although this study attributed phos-phorylation to ATM and/or ATR, given that DNA-PK alsophosphorylates SQ/TQ sites, some of these sites could equally re-present DNA-PK-dependent phosphorylation events. Regardless



of what its physiological substrates are, the ability of small-molecule inhibitors of DNA-PKcs to radiosensitize cells suggeststhat DNA-PK may be an attractive therapeutic target as a radiationsensitizer (reviewed in [60,171,172]).

As discussed above, IR-induced DNA damage is highly com-plex, and produces damage to bases and production of SSBs aswell as DSBs. Indeed, the number of damaged bases and SSBs faroutweighs the number of DSBs produced by IR [6]. It is thereforelikely that BER- and SSB-repair pathways must function in closeproximity to DSB-repair pathways. Interestingly, several potentialconnections between the proteins involved in BER, SSB repair andNHEJ are beginning to emerge. For example, PNK and APLFinteract not only with XRCC4 [109,113,114,116,117], but alsowith XRCC1, a protein required for both BER and SSB repair[113,173,174]. It is also interesting to note that XRCC1 has beenidentified as an IR-inducible target of DNA-PK [175]. Proteinsinvolved in BER and SSB repair have also been implicated inalternative DSB-repair pathways [13–16]. Thus it seems likelythat NHEJ-, BER- and SSB-repair pathways function in a co-ordinated manner to repair IR-induced DNA damage.

Another outstanding question in the field is how a cell decides torepair DSBs via NHEJ, HDR or alternative end-joining pathways.This area is currently the topic of intense study [2,11,176,177].One potential mechanism for determining pathway choiceappears to be via cell-cycle-regulated expression of the CtIP[CtBP (C-terminus-binding protein)-interacting protein]/Sae2(SUMO-activating enzyme subunit 2) protein, which stimulatesresection at the DSB by the MRN (Mre11–Rad50–Nbs1)complex, thus promoting HDR in S- and G2-phases of thecell cycle (reviewed in [178]). Another critical, yet unresolved,question regarding pathway choice is what regulates recognitionof DSBs by Ku compared with the MRN complex, which isrequired not only for initiation of HDR, but also for activationof ATM-dependent signalling pathways [165–167]. Finally, wenote that ATM phosphorylates several NHEJ proteins, includingArtemis [94] and XLF [59], as well as DNA-PKcs ([71] and P.Douglas and S. P. Lees-Miller, unpublished work), suggestingthat the PIKK family members act together to co-ordinate theDNA-damage response rather than in separate pathways.

Understanding how these multiple pathways are connectedand regulated both temporally and spatially will provide criticalinsights into the mechanisms by which cells deal with the delet-erious effects of DNA damage and prevent genomic instability.

ACKNOWLEDGEMENTS

We thank T. Beattie, E. Kurz, M. Weinfeld and members of the Lees-Miller laboratory forhelpful comments and suggestions.

FUNDING

Work in the S. P. L.-M. laboratory is supported by the Canadian Institutes of Health Research[grant number 13639]. S. P. L.-M. is a Scientist of the Alberta Heritage Foundation forMedical Research and holds the Engineered Air Chair in Cancer Research. B. L. M. issupported by studentships from the Alberta Heritage Foundation for Medical Researchand the Natural Sciences and Engineering Research Council of Canada.

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Received 27 February 2008/13 October 2008; accepted 14 October 2008Published on the Internet 16 January 2009, doi:10.1042/BJ20080413


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