The role of the non-homologous

end-joining pathway in lymphocyte

development

Sean Rooney

Jayanta Chaudhuri

Frederick W. Alt

Authors’ address

Sean Rooney, Jayanta Chaudhuri, Frederick W. Alt,

Howard Hughes Medical Institute, The

Children’s Hospital, The Department of

Genetics, Harvard Medical School and The

Center for Blood Research, Boston, MA, USA.

Correspondence to:

Fred W. Alt

Children’s Hospital

300 Longwood Avenue

New Research Building, 9th Floor

Boston, MA 02115, USA

Tel.: þ1 617 919 2539

E-mail: alt@enders.tch.harvard.edu

Acknowledgements

This work was supported by grants from the NIH

(AI35714, AI20047, and AI31541) and NCI (CA92625).

F.W.A. is an investigator and J.C. an associate of the

Howard Hughes Medical Institute. We apologize to those

whose work we could not cite due to space constraints.

Summary: One of the most toxic insults a cell can incur is a disruption ofits linear DNA in the form of a double-strand break (DSB). Left unrepaired,or repaired improperly, these lesions can result in cell death or neoplastictransformation. Despite these dangers, lymphoid cells purposely introduceDSBs into their genome to maximize the diversity and effector functions oftheir antigen receptor genes. While the generation of breaks requiresdistinct lymphoid-specific factors, their resolution requires variousubiquitously expressed DNA-repair proteins, known collectively as thenon-homologous end-joining pathway. In this review, we discuss thefactors that constitute this pathway as well as the evidence of their involve-ment in two lymphoid-specific DNA recombination events.

Introduction

The adaptive arm of the immune system is predicated upon

the recognition of foreign antigen by immunoglobulin (Ig)

and/or T-cell receptors (TCRs) expressed on the surface of B

and T lymphocytes, respectively. While the number of anti-

gens is seemingly limitless, the mammalian genome contains

only a finite number of antigen recognition genes. This prob-

lem is dealt with in a novel fashion: the reorganization of

germline DNA segments into unique antigen receptor genes, a

process referred to as V(D)J recombination (1).

V(D)J recombination is a site-specific event that takes place

at six distinct loci: TCRb, g, a/d loci, Ig heavy chain (IgH), and

k or l light chain (LC) loci. Recombination occurs between

component variable (V), junctional (J), and, in some cases,

diversity (D) gene segments, with fused VJ or VDJ coding

sequence subsequently joined to a constant region segment

through RNA splicing. Because most antigen loci have numer-

ous gene segments, a significant level of antigen-receptor

diversity is generated solely through multiple combinations

of fused products (2).

However, assortment of germline V gene segments alone

is not sufficient to account for the wide range of antigen

Immunological Reviews 2004

Vol. 200: 115–131

Printed in Denmark. All rights reserved

Copyright � Blackwell Munksgaard 2004

Immunological Reviews0105-2896

115

specificities. For example, the sequence of Va and Vb gene

segments is highly restricted for two of the three domains

responsible for antigen recognition [complementarity-deter-

mining region (CDR) 1 and CDR2] (3). By comparison,

CDR3, which spans the junction of fused gene segments, is

highly variable. Much of this junctional diversity is a result

of the imprecise repair of DNA double-strand breaks (DSBs)

introduced at specific sites within antigen-receptor gene

segments during the V(D)J reaction.

The V(D)J recombination reaction occurs in two distinct

steps. First, the lymphoid-specific endonuclease recombinase-

activating genes 1 and 2 (RAG-1 and RAG-2, collectively RAG)

introduce DSBs precisely between gene segments undergoing

recombination and their flanking recombination signal

sequences (RSSs) (4) (Fig. 1A). The RAG proteins generate

breaks by nicking a single DNA strand between the coding

flank and the adjacent RSS, leaving a 30 hydroxyl group on the

coding side and a 50 phosphoryl group on the RSS side. The

30 hydroxyl group then attacks the phosphodiester backbone of

the opposing strand, resulting in a covalently sealed hairpin

coding end and in a blunt 50 phosphorylated recombination

signal (RS) end (5).

The second half of the V(D)J reaction involves the resolution

of RAG-generated DSBs. Whereas the RS ends are fused directly

to form RS joins, coding-end hairpins must be opened and

processed prior to coding join formation (Fig. 1B). Hairpin

opening in vivo usually occurs at or within several nucleotides

of the apex (6, 7). Opening away from the apex results in

over-hanging nucleotides, which, if incorporated into the

join, generate non-germline palindromic (P)-nucleotide add-

itions (8, 9). Coding joins are further diversified through the

addition of non-templated (N)-nucleotides by the lymphoid-

specific terminal deoxynucleotidyl transferase (10–12) or

through the deletion of a small number of nucleotides from

coding sequences. Although the two distinct intermediates

generated by RAG cleavage must be dealt with in a different

manner, the repair of each is dependent upon the same ubi-

quitously expressed set of DNA-repair factors, collectively

referred to as the non-homologous end-joining (NHEJ)

pathway.

In eukaryotes, a majority of DSBs are resolved through two

repair pathways. Homologous recombination (HR) uses infor-

mation from a homologous template to accurately repair

breaks, and it is generally limited in mammalian cells to late

S-phase and G2-phase of the cell cycle, when sister chromatids

present readily available templates (13). NHEJ rejoins broken

DNA ends with little or no sequence homology, and it is the

predominant pathway for repair during G1 (14, 15), the stage

at which RAG-associated DSBs are generated (16).

The NHEJ pathway consists of at least six proteins. The

DNA-binding subunits Ku70 and Ku80, together with the

DNA-dependent protein kinase catalytic subunit (DNA-PKcs),

form the DNA-PK holoenzyme (DNA-PK), believed to be

involved early in the recognition of DSBs (17). The nuclease

Artemis forms a complex with DNA-PKcs, and it is responsible

for processing hairpin ends prior to their ligation. DNA ligase

RAG

Nicking

Nucleophilic attack

Double-strand break

HO

HO

HO

OH

OH

OH

Ku70/80

DNA-PKcsArtemis

Ligase IV/XRCC4(DNA-PKcs)

Ligase IV/XRCC4TdT

Precise RS join

Imprecise coding join

A B

Fig. 1. The V(D)J reaction. (A) Introductionof double-strand breaks by the recombinase-activating gene (RAG) endonuclease. (B) Thenon-homologous end-joining (NHEJ) factorsresponsible for recombination signal (RS)and coding joining.

Rooney et al � The NHEJ pathway in lymphocyte development

116 Immunological Reviews 200/2004

IV, together with its partner XRCC4, catalyzes the final step of

the reaction. In this review, we summarize the role of each

factor, with particular attention paid to the impact of its

absence on V(D)J recombination and lymphocyte develop-

ment. We also discuss the potential role of NHEJ in a second

lymphoid-specific DNA recombination event, known as IgHC

class switch recombination (CSR). Finally, we summarize the

evidence that defects in NHEJ promote aberrant recombination

events, which may ultimately lead to lymphomagenesis.

The NHEJ factors and V(D)J recombination

Ku70, Ku80, DNA-PKcs, ligase IV, and XRCC4 were each

linked to DSB repair and V(D)J recombination, either directly

or indirectly, through studies of ionizing-radiation (IR)-

sensitive Chinese hamster ovary (CHO) cell lines as well as

of the naturally occurring severe combined immunodeficiency

(SCID) mice (18, 19). Artemis was subsequently identified as

being mutated in a subset of human SCID patients (20). The

NHEJ pathway is partially conserved in yeast (21), and it may

also play a role in DSB repair in prokaryotes (22). However,

while the yeast homologs of Ku70, Ku80, XRCC4, and ligase IV

have all been identified, DNA-PKcs and, potentially, Artemis

appear restricted to higher eukaryotes (23).

The DNA-PK holoenzyme

DNA-PK was first characterized from cell extracts as a kinase

specifically activated by double-stranded DNA but not by

single-stranded DNA or RNA (24–26). Subsequent work

determined that the DNA-PK holoenzyme is a nuclear serine/

threonine kinase (27), consisting of the DNA-binding Ku

heterodimer and the DNA-PKcs (28–30).

Ku was originally identified as an autoantigen from

scleroderma–polymyositis-overlap syndrome patients (31).

Ku is a DNA-binding heterodimer, consisting of two tightly

associated subunits, Ku70 and Ku80 (sometimes referred to

as Ku86), which binds with high affinity to free double-

stranded DNA ends, DNA hairpins, single-strand nicks, or

gaps in a sequence-independent manner (32). A role for Ku

in DSB repair was demonstrated by the ability of human

Ku86 to complement the IR-sensitivity and V(D)J recom-

bination defects of xrs6 CHO cells (33–35), and subsequently

verified through gene-targeted mutation of each subunit in

mice (36, 37). The recognition of DNA ends by the Ku

heterodimer is thought to be an early event in NHEJ (38)

and may be responsible for directing repair away from HR

(39–41).

Binding of Ku to DNA is dependent upon heterodimeriza-

tion, as neither subunit can bind DNA alone (42–45). Inter-

action of the heterodimer is mediated through amino acids

449–578 of Ku70 and amino acids 439–592 of Ku80 (46),

and these two regions share significant sequence homology

(46), suggesting that the two subunits of Ku arose through a

gene-duplication event (47). Consistent with this idea, a single

DNA-binding protein with significant homology to Ku has

been identified in prokaryotes (22). Ku70 and Ku80 together

form a ring structure with a central channel large enough to

accommodate a single double-stranded DNA molecule (48).

The heterodimer does not make contact with a single DNA

base, and it has only limited contact with the sugar-phosphate

backbone, in agreement with the lack of sequence-specific

binding for Ku. However, the ring structure fits well into

both the major and minor groove of duplex DNA, orientating

the DNA in a particular plane, a feature that may be relevant

for aligning DNA ends prior to their ligation (49, 50).

Upon binding to DNA, a 12-amino-acid sequence at the

C-terminus of Ku80 is exposed and recruits DNA-PKcs (46,

51). This, in turn, stimulates the inward translocation of Ku,

allowing DNA-PKcs to bind directly to DNA ends (52, 53). At

470 kDa, DNA-PKcs is one of the largest known cellular pro-

teins (47). Despite its large size, the only clearly defined motif

within the subunit is a C-terminal phosphatidylinositol

3-kinase (PI3K) domain (54, 55), characteristic of kinases

with lipid substrates (56). However, DNA-PKcs is specific

for protein substrates, preferentially phosphorylating S/T-Q

motifs (serine or threonine followed by a glutamine) (27, 57,

58). As a result, DNA-PKcs is categorized as a member of the

PI3K-related protein kinase (PIKK) family, which includes the

cell-cycle checkpoint proteins antaxia telangiectasia mutated

(ATM) and ATM- and RAD3-related (ATR) (59).

The role of DNA-PKcs in V(D)J recombination was first

appreciated through studies of the IR-sensitive SCID mouse

and transient transfecion of V3 CHO cell line. Both V3 cells

and SCID mice have a specific defect in coding with RAG and

V(D)J recombination subtrates join formation, while RS join

formation is relatively unaffected (60–63). Furthermore, the

rare coding joins recovered from both SCID and V3 cells have

elevated levels of P-nucleotide additions, indicating that RAG-

generated hairpins are opened aberrantly (8). While the V3

defect results from the deletion of prkdc (the gene encoding

DNA-PKcs) (64), SCID mice harbor a single point mutation at

amino acid 4046 of DNA-PKcs, resulting in a truncated protein

(65–67). The truncation leaves the kinase domain intact;

however, the deleted region is highly conserved among PIKK

family members (66), and the loss of this domain greatly

Rooney et al � The NHEJ pathway in lymphocyte development

Immunological Reviews 200/2004 117

reduces the kinase activity of DNA-PKcs in vitro (68). The

requirement for DNA-PKcs kinase activity in V(D)J recombin-

ation was further demonstrated by the inability of a kinase-

dead DNA-PKcs to complement the coding join defect of V3

cells (69). However, it was only with the identification of the

nuclease Artemis (20) that the ability of DNA-PKcs to regulate

coding join formation was fully understood (see below).

Moreover, autophosphorylation of DNA-PKcs appears to be

important for proper end processing. DNA-PKcs has seven

different sites that undergo phosphorylation in vivo, six of

which are clustered in a 38-amino-acid region in the middle

of the protein (70). This modification appears relevant to

NHEJ in vivo, as mutation of the sites within this cluster

abrogates the ability of DNA-PKcs to rescue the IR sensitivity

of V3 cells (71, 72), without affecting the kinase activity of

DNA-PKcs in vitro (72). A recent report suggests that auto-

phosphorylation occurs following the synapse of two DNA-

PK-bound DNA ends, possibly resulting in a conformational

change of the holoenzyme that facilitates further processing

and/or ligation of DNA ends (73).

Several studies have demonstrated that DNA-PKcs is also

necessary for the proper repair of blunt-ended breaks (74–77),

indicating that the holoenzyme has a role in NHEJ beyond

processing incompatible DNA ends. DNA-PK may serve as a

scaffold for recruiting other factors necessary for DSB repair,

such as the XRCC4–ligase IV complex (78–82). DNA-PK has

also been shown to interact with multiple DNA molecules

simultaneously (83, 84), leading to the speculation that the

holoenzyme may mediate the synapse of DNA ends. Neither

the recruitment of the XRCC4–ligase IV complex nor the

synapse formation requires the kinase activity of DNA-PKcs

(81, 84). Together, these data suggest that kinase-dead ver-

sions of DNA-PKcs, such as the murine SCID mutation, retain

partial function. In support of this notion, it has been recently

reported that the kinase activity of DNA-PKcs, but not DNA-

PKcs itself, is expendable for transposition of the sleeping

beauty element (85).

Ku70, Ku80, and DNA-PKcs have all been inactivated in

mice by gene targeting, and deletion of each results in defect-

ive T- and B-cell development, due to the inability to repair

RAG-induced DSBs (37, 86–92). Covalently sealed coding

ends accumulate in thymi of these mice (86, 88, 92), demon-

strating that the holoenzyme is employed for hairpin opening

in vivo. Furthermore, coding join formation is greatly reduced

in fibroblasts isolated from each of these mice, and the rare

joins that are recovered display large deletions of the coding

flanks, indicating that the coding join formation that does

occur is grossly aberrant (86, 92, 93). Moreover, it is worth

emphasizing that coding joins isolated from these cells,

including DNA-PKcs-null fibroblasts, do not display the ele-

vated levels of P-nucleotide additions that characterize joins

from kinase-dead SCID mice. Therefore, coding join formation

in the absence of the kinase activity may be distinct from what

occurs in the absence of the entire protein, supporting the idea

that DNA-PKcs has multiple functions in this process.

The frequency and fidelity of RS joining is also defective in

the absence of each subunit of the DNA-PK holoenzyme, albeit

more severely so for Ku than for DNA-PKcs. The level of RS

joining is greatly reduced in Ku-deficient fibroblasts assayed

by transient transfection of a V(D)J recombination substrate.

Furthermore, the rare RS joins recovered from these cells

exhibit deletions and regions of microhomology (86). It has

been suggested that the Ku heterodimer facilitates alignment

of DNA ends prior to ligation (49, 50) and thus may obviate

the need for such microhomology in normal RS joining. SCID

cells have little or no impairment in the level of RS joining and

most are precise (60, 61). However, DNA-PKcs-null fibro-

blasts have a more variable defect in both the level and the

precision of RS joining, although a number of joins remain

precise (93, 94). While more studies need to be performed,

the potential differences in the frequency and the fidelity of RS

joining between SCID and DNA-PKcs-deficient cells further

support the notion that the SCID mutation is not equivalent

to the complete loss of DNA-PKcs.

Consistent with Ku having functions beyond the context of

the DNA-PK holoenzyme, the overall phenotypic effects of

DNA-PKcs deficiency in mice are clearly not equivalent to

the loss of the Ku heterodimer. Aside from the defects in

lymphocyte development, the only obvious phenotype of

DNA-PKcs-null mice is an increased level of organismal and

cellular sensitivity to IR, consistent with a general defect in

DSB repair (89, 90, 92, 95). Ku-deficient mice have increased

levels of neuronal apoptosis (96), are small (approximately

50% by body weight) compared to their littermates (37, 86,

87), and incur age-specific changes significantly earlier than

controls (97). Likewise, fibroblasts isolated from Ku-deficient

mice display growth defects, premature cellular senescence,

and hypersensitivity to IR, generally greater than that of DNA-

PKcs-deficient cells (37, 86). Inactivation of the cell-cycle

checkpoint protein p53 suppresses the growth defects of

Ku-deficient cells (98, 99); however, it does not rescue the

dwarfism observed in these mice.

Finally, inactivation of Ku70, Ku80, or DNA-PKcs in the

context of ATM-deficiency is synergistically lethal at a very

early embryonic stage (100, 101), suggesting that these two

PIKK kinases may play overlapping roles in development. Such

Rooney et al � The NHEJ pathway in lymphocyte development

118 Immunological Reviews 200/2004

potential overlapping roles likely do not involve NHEJ, as

ATM deficiency actually suppresses the embryonic lethality

associated with XRCC4- or ligase IV-deficient mice (101). In

this regard, both Ku and DNA-PKcs appear to have other

functions not directly related to NHEJ, including telomere main-

tenance (102), phosphorylation of the histone variant H2AX

(103–106), regulation of the WRN nuclease (107–109), DNA

replication (110, 111), cell signaling (112, 113), or regulating

apoptosis (114, 115).

Artemis

Approximately 20% of human SCID patients are characterized

by a complete absence of mature T and B cells, despite normal

natural killer (NK)-cell development (T-B-SCID) (116). Muta-

tions in rag1 or in rag2 have been observed in a majority of

these patients, and fibroblasts from these patients display

normal radiosensitivity. However, fibroblasts from a subset

of these patients are highly radiosensitive (RS-SCID) (117).

V(D)J recombination is defective in these patients, as evi-

denced by grossly abnormal D-to-J rearrangements of the

IgH locus (118). Furthermore, RS-SCID fibroblasts have a

specific defect in coding join formation, while RS join forma-

tion appears unaffected (119). While this profile is similar to

that of SCID mice, RS-SCID fibroblasts have normal DNA-PKcs

kinase activity and normal Ku DNA-binding activity (119,

120). Genetic analysis localized the rs-scid mutation to a region

on chromosome 10 (121), associated with the high incidence

of T-B-SCID observed in Athabascan-speaking Native Ameri-

cans (SCIDA) (122). The rs-scid gene was cloned and was

found to encode a novel 77.6 kDa protein named Artemis

(20). Similar defects in Artemis have subsequently been

shown to underlie related human SCID cohorts (123–125).

Artemis is a member of the metallo-b-lactamase superfamily

of proteins (20), enzymes that use co-ordinated zinc(II) ions

to activate water molecules for the hydrolysis of covalent

bonds (126). First identified in prokaryotes based on the

ability to inactivate bactericidal compounds, b-lactamases

have evolved to recognize a wide variety of substrates (127).

While having little primary homology, metallo-b-lactamases

share a characteristic secondary feature, known as a metallo-b-lactamase fold (126). Artemis has been further classified into a

subfamily of metallo-b-lactamases that act on nucleic acid

substrates and share a second highly conserved motif, referred

to as the b-CASP domain (128).

The homology of Artemis with enzymes that catalyze the

hydrolysis of covalent bonds, as well as the parallel phenotypes

of RS-SCID patients and SCID mice, led to the speculation that

coding-end processing might be mediated by DNA-PKcs

through the regulation of Artemis (20). Biochemical analysis

strongly supported such a model, as DNA-PKcs forms a com-

plex with Artemis in vivo and phosphorylates Artemis in vitro

(129). Furthermore, a DNA-PKcs–Artemis complex preferen-

tially opens RAG-generated hairpins two nucleotides 30 of the

apex in vitro (129), similar to what has been observed in vivo

(6, 7). DNA-PKcs also alters the nuclease activity of Artemis

from an intrinsic single-strand-specific 50-to-30 exonuclease to

an endonuclease that works on both 50 and 30 overhangs

(129), an activity that may be relevant to the role of Artemis

in general DSB repair.

Like DNA-PKcs-null mice, Artemis-deficient (ArtN/N) mice

appear relatively normal, with a defective lymphocyte devel-

opment as the only obvious phenotype (93). The block in

lymphocyte development observed in ArtN/N mice is due to a

specific defect in coding join formation, and covalently sealed

hairpin intermediates can be detected in thymi of ArtN/N mice,

validating Artemis as a nuclease responsible for processing

coding ends in vivo. However, Artemis-deficient mice display

a variable level of leaky CD4þ T-cell development, and coding

joins can be recovered from Artemis-deficient cell lines, albeit

at greatly reduced levels (93, 130). Similar to in SCID mice,

the joins recovered in the absence of Artemis are characterized

by an increase in the size and frequency of P-nucleotide add-

ition, indicating that the little coding join formation that does

occur is aberrant.

Unlike what has been observed for a single RS-SCID infant

(131), it does not appear that maternal CD4þ T cells contri-

bute to the leaky T-cell development in ArtN/N mice (Rooney

and Alt, unpublished data). Instead, it seems more likely that

an alternative factor(s) is capable of opening hairpins in the

absence of Artemis. Several other proteins have been shown to

possess hairpin-opening activity in vitro, including the Mre11–

Rad50–Nbs1 complex (132) and the RAG proteins (133,

134). While the hairpin-opening activities of both have char-

acteristics that are inconsistent with normal coding-end pro-

cessing in vivo (9), these or other factors still may be able to

compensate for the loss of Artemis at a low level.

While both DNA-PKcs and Artemis-deficient fibroblasts are

clearly more sensitive to IR than wildtype, they are not as

sensitive as Ku70-, Ku80-, XRCC4-, or ligase IV-deficient

fibroblasts (92, 93). Similarly, mice deficient in DNA-PKcs

or Artemis lack the more severe phenotypes observed in other

NHEJ-deficient mice. These findings support the idea that the

DNA-PKcs–Artemis complex might have arisen later in evolu-

tion to deal with a subset of DSBs that required endonucleo-

lytic processing, such as those with staggered or damaged ends

Rooney et al � The NHEJ pathway in lymphocyte development

Immunological Reviews 200/2004 119

(23, 93, 130). However, in the context of general DSB repair,

it remains to be determined exactly what types of ends require

processing by Artemis and how this function overlaps with

other nucleases implicated in this process, such as WRN, FEN-1,

or the Mre11 complex (135–137).

The XRCC4–ligase IV complex

Following the processing and alignment of DNA ends, V(D)J

recombination appears to be completed via the ligation of two

coding ends or two RS ends by the XRCC4–ligase IV complex.

Xrcc4 was identified through complementation of the IR sensi-

tivity of XR-1 CHO cells (138), and encodes a 326-amino-acid

product with no obvious homology with any known proteins.

XRCC4 was subsequently found to co-purify with DNA ligase

IV (139, 140), and the role of this complex in both NHEJ and

V(D)J recombination was validated through gene-targeted

mutation in mice (141, 142).

The interaction of ligase IV with XRCC4 is thought to

stimulate ligase IV activity in several ways. Ligase IV is severely

reduced in XR-1 cells (143), suggesting that XRCC4 stabilizes

ligase IV protein expression. Like Ku, XRCC4 may have a role

in aligning DSBs prior to their ligation (50, 80, 144, 145).

Moreover, XRCC4 can stimulate the adenylation of lysine

residues within the catalytic core of ligase IV, the first step in

the formation of a new phosphodiester bond (145). In this

context, human patients have been identified with reduced

levels of ligase IV activity (146, 147), and the mutations

underlying this defect prevent either the interaction with

XRCC4 (146) or the adenylation or the ligase IV active site

(147).

Although XRCC4 itself has been reported to have DNA end-

binding activity (145), the XRCC4–ligase IV complex most

likely is recruited to DNA ends through interactions with the

DNA-PK holoenzyme (78–81). In this regard, the C-terminus

of ligase IV has two BRCT (BRCA-1 C-terminal) domains, a

motif found in several DNA-repair proteins and recently

shown to be phosphoserine or phosphothreonine-specific-

binding module (148–150). It is interesting to speculate that

these repeats may be involved in the interaction between

DNA-PK and the XRCC4–ligase IV complex, possibly through

the binding of autophosphorylation sites on DNA-PKcs.

As mentioned, gene-targeted mutations of LIGASE IV or

XRCC4 have verified a role for both in V(D)J recombination.

While homozygous mutations of either gene result in embryo-

nic lethality, XRCC4- and ligase IV-deficient embryos exhibit

a complete block in lymphoid development (141, 142), and

IgH rearrangements cloned from ligase IV-deficient progenitor

B cells grown in culture are grossly aberrant (141). Further-

more, fibroblasts from these mice fail to support either coding

join or RS join formation by transient transfection (141, 142).

In addition to V(D)J recombination defects, XRCC4- and

ligase IV-deficient mice share a number of other phenotypes,

indicative of the absolute requirement for this complex in

NHEJ. XRCC4- and ligase IV-deficient mice generally die in

utero by day E16.5. Although the exact cause of death has not

been established, these embryos display massive apoptosis of

post-mitotic neurons in the central nervous system (142, 151,

152). It remains unclear why these cells, in particular, are

sensitive to DSB repair defects (142), given that the embryonic

lethality and neuronal apoptosis can be suppressed by inactiva-

tion of p53 or ATM (101, 153–155), it appears that both are

the result of cell-cycle checkpoint responses to the persistence

of unresolved DSBs, rather than the inability to repair DSBs per

se. However, inactivation of p53 does not rescue NHEJ per se,

therefore, lymphoid development remains defective in these

mice (153, 154), as progenitor cells lack the functional anti-

gen receptors required to promote further differentiation

(156, 157). Similarly, fibroblasts isolated from XRCC4- or

ligase IV-deficient mice exhibit substantial growth defects

and premature cellular senescence, as well as hypersensitivity

to IR (141, 142).

Finally, the embryonic lethality of ligase IV-deficient mice

can also be suppressed by inactivation of Ku80 (158). Similar

observations have been reported in a chicken pre-B-cell line

(39, 40), and together these data support a model where the

binding of the Ku heterodimer to DSBs prevents repair by

another pathway, such as HR, even in the absence of func-

tional NHEJ (39–41). Alternatively, it has been proposed that

the Ku facilitates a critical loss of DNA through the serial

recruitment of the DNA-PKcs–Artemis nucleolytic complex to

ends left unrepaired in the absence of ligase IV (158). In this

context, it remains to be determined whether inactivation of

DNA-PKcs or Artemis also rescues the embryonic lethality

associated with ligase IV deficiencies.

The role of NHEJ in IgH CSR

Upon migration to peripheral lymphoid organs, mature B

lymphocytes can be stimulated to undergo a second DNA

recombination event. In the appropriate context, B cells switch

the effector function of their IgH through the replacement of

the m constant-gene exons (Cm) with one of several sets of

downstream constant-region gene exons (referred to as CHgenes) (Fig. 2), while retaining the specificity of their variable

region. This process is referred to as IgH CSR (159).

Rooney et al � The NHEJ pathway in lymphocyte development

120 Immunological Reviews 200/2004

Unlike V(D)J recombination, CSR does not require the RAG

proteins or short, canonical RSSs (160). Instead, CSR as well as

the related process of somatic hypermutation (SHM) is depen-

dent upon activation-induced deaminase (AID) (161), which

converts cytidine to uridine in the context of single-stranded

DNA substrates (162–165). Germline transcription through

CH genes stimulated to undergo recombination is also

required, apparently to generate single-stranded DNA sub-

strates for AID (163), and disruption of cis-acting elements

promoting transcription of a particular CH gene blocks switch-

ing to that isotype (159).

With the exception of Cd (which switches through normal

RNA splicing), each CH gene is organized into a discrete unit

comprised of a 50 promoter region, an I exon, a highly

repetitive, G/C-rich element referred to as the switch region

(S region), and finally the CH exons. Transcription of S

regions, both in vivo and in vitro, has been shown to result in

the formation of a stable RNA : DNA hybrid between the

switch transcript and the C-rich template strand, displacing

the non-template G-strand in an R-loop structure (166, 167).

Inversion of S regions changes the template strand from C-rich

to G-rich and severely inhibits both R-loop formation in vitro

(166, 167) and CSR in vivo (168). Furthermore, a synthetic

sequence, unrelated to S regions but capable of forming R

loops in vitro, supports CSR in vivo (168). Additionally, in vitro

studies suggest that transcribed S regions may also assume

secondary structures, such as stem-loops resulting from the

high density of palindromic sequence within the S region or

four-stranded G-quartets (G4 DNA) stabilized by base pairing

between G residues (169, 170).

These observations provide an important link between the

requirement for germline transcription and the activity of AID

in CSR, and they have led to the following model: transcrip-

tion through S regions induces the formation of single-

stranded DNA that serves as a substrate for AID (163). The

generation of stable R loops may be one means to generate

such single-stranded DNA structures, although other mechan-

isms must exist (168, 171). AID-mediated deamination of

cytidine to uridine results in a dU/dG mismatch, which then

serves as a substrate for either the base excision repair (BER) or

the mismatch repair (MMR) pathways, both of which generate

a single-strand nick or gap at the site of the mismatch (172).

Deamination of the proximal cytidine bases on opposing

strands would result in a staggered DSB within S regions

(Fig. 3). Consistent with such a model, mutations of either

BER or MMR factors alter CSR in mice (173–179).

There is significant evidence in support of CSR proceeding

through a DSB intermediate. First, CSR between two S regions

results in the deletion of the intervening sequence (180), and the

excised products can be detected as extrachromosomal circles

following CSR (181–183). Second, DSBs in S regions of B cells

stimulated to undergo CSR can be detected by ligation-mediated

polymerase chain reaction (LM-PCR) (184), and these breaks

appear to be AID dependent (185). Finally, g-H2AX foci, which

form at sites of DNA damage, associate with the IgH locus of

activated B cells in an AID-dependent manner (186). However, a

word of caution is needed. LM-PCR also detects DSBs in V genes

in an AID-independent manner (187–189), and g-H2AX foci also

can be found in V regions following induction of AID (190).

Therefore, while it appears quite likely that DSBs are an integral

part of the CSR mechanism, it remains to be formally proven.

Allowing for CSR-associated DSBs, several studies suggest

that the repair of these breaks may involve a subset of the NHEJ

factors. Switch junctions cloned from wildtype B cells display

little, if any, homology (191), demonstrating that HR is not

involved in this process. Moreover, in studies of mature B cells

Class switch product Excised circle

IgG2a

DNA-PKcsNHEJ?H2AX

Double-strand break

UNG/APE1

AID

R-loop

Cγ2a

γ3 γ1

γ2bµδ

u u u u u

c c

Sµ

Cµ Cγ2a

Cγ2a

Cγ2a

Cγ2a

Cµ

Cµ

Cµ

µ δ γ 3 γ 1 γ 2b γ 2a ε α

c c c

IgM

A

B

c

u

Sγ2a

Fig. 2. Immunoglobulin (Ig) heavy chain (HC) class switchrecombination (CSR). (A) Germline organization of the IgH constantregions. (B) Model for the generation of activation-induced deaminase(AID)-dependent double-strand breaks during CSR.

Rooney et al � The NHEJ pathway in lymphocyte development

Immunological Reviews 200/2004 121

reconstituted by IgH and LC knock-in alleles, inactivation of

certain NHEJ factors dramatically reduces CSR. Both Ku70- and

Ku80-deficient B cells are unable to undergo CSR either in vivo

or in vitro (192, 193). However, as deletion of either Ku70 or

Ku80 results in a general cellular proliferation defect (37, 86),

it is unclear whether Ku-deficient B cells are defective in CSR

per se or simply die due to other causes during the proliferative

stage of B-cell activation prior to CSR. While the finding that a

subset of Ku80-deficient B cells that undergo multiple of the

former rounds of cell division still fail to switch is strongly

suggestive (194), it is possible that this population represents

cells that were not fully activated for CSR.

Like Ku70/80, DNA-PKcs appears to be involved, either

directly or indirectly, in CSR, as deletion of DNA-PKcs blocks

CSR to all isotypes except for IgG1 (193). Unlike Ku-deficient

cells, this effect appears to be specific as DNA-PKcs-null B cells

proliferate normally in vitro and switching is not rescued by

crossing p53 deficiency (193). By comparison, B cells from

SCID mice appear to undergo CSR to all IgH isotypes, albeit at

reduced levels (195, 196). While differences in strain back-

ground and specificity of the knock-in Ig receptor must be

accounted for, the difference in CSR observed in DNA-PKcs-

null and SCID mice may reflect the complete loss of function in

the former and partial loss of function in the latter. In this case,

activities of DNA-PKcs not directly related to its kinase activity

may be those relevant to CSR.

The role of DNA-PK in CSR is clearly different from its role

in V(D)J recombination. Whereas coding end hairpins must

Chromosome 12

D V

RAG

DSB replication

Invasion

Chromosome 15

Random breakage

C12;15 translocation

Replication

Replication

Breakage

Fusion

Telomere donation fromthird chromosome

Complicon with co-amplified IgH and c-myc

Breakage-fusion-bridge cycles

c-myc

J

J

J

Fig. 3. Breakage–fusion–bridge cycle

promotes lymphogenesis in the absenceof proper double-strand break repair and

functional cell-cycle checkpoints. Modeldetailing the molecular events leading toco-amplification of IgH/c-myc in NHEJ/p53pro-B cell tumors is mediated throughbreakage–fusion–bridge cycle.

Rooney et al � The NHEJ pathway in lymphocyte development

122 Immunological Reviews 200/2004

be processed through the co-ordinate activity of DNA-PKcs

and Artemis, CSR occurs at wildtype levels in the absence of

Artemis (Rooney, Manis, and Alt, in preparation). Therefore,

processing of DSBs by Artemis is not required for CSR, despite

evidence that these breaks have staggered ends (197). It is

possible that staggered breaks are not the only relevant DSB in

CSR, that a CSR-specific nuclease is required to process these

ends (198, 199), or that CSR-associated breaks are repaired by

a different pathway. Additionally, given that both Ku and

DNA-PKcs have functions that lie beyond NHEJ (101), the

impact of mutations on these factors, while specific for CSR,

cannot be taken as absolute evidence for a role of the NHEJ

pathway in this process. Therefore, examination of CSR in the

absence of XRCC4 or ligase IV, the two factors thought to be

exclusively involved in all NHEJ repair, is needed to directly

assess the relevance of this pathway to CSR.

Several factors outside of NHEJ have been implicated in the

repair of CSR-associated breaks. In particular, H2AX-deficient

mice are defective for CSR (186); however, intra-switch-

region deletions (ISRs), a related AID-dependent process

(200), remain intact (194). H2AX is phosphorylated

(g-H2AX) in response to DNA damage, and subsequently, it

recruits a number of different DNA-repair factors to the site

of DSBs. g-H2AX has also been proposed to facilitate repair

by anchoring broken DNA ends in proximity through its

interaction with several other factors including 53BP1, the

Mre11–Rad50–Nbs1 complex, and MDC1 (201). Similarly,

H2AX may be required to ensure the long-range interaction

of two S regions necessary for proper CSR (194, 201). Con-

sistent with a role for H2AX in CSR, H2AX haploinsuffi-

ciency, in the context of p53 deficiency, results in the

appearance of B-cell lymphomas with translocations invol-

ving IgH S regions (202). Moreover, loss of the DNA damage

response factor 53BP1 also severely impairs CSR, further

supporting the notion that H2AX and 53BP1 may work

together, as well as with other damage response factors, in

the joining phase of the CSR (Manis et al., submitted for

publication).

The exact timing of AID-induced DSBs remains unresolved.

Single-strand mutations accumulate during G1 in a tumor cell

line stimulated to undergo SHM (189). Similarly, AID-

dependent g-H2AX foci are recruited to switch regions in

activated B cells during G1/early S phase (186). However, it

remains possible that in the case of CSR, AID introduces

mutations on a single DNA strand in G1, and following the

activity of the BER/MMR pathway(s), a single-strand nick is

converted to a DSB by DNA replication of the IgH locus in S

phase. Such a model would be in agreement with the observa-

tion that CSR requires cell division (203–205) and is blocked

by DNA replication inhibitors. As the choice of DSB repair

pathways can be greatly influenced by the stage of the cell

cycle that breaks occur (14, 15), determining the exact timing

of AID-induced breaks remains quite important.

The consequences of defective DSB repair in

lymphocytes

The importance of proper DNA maintenance is underscored

by the observation that inactivation of DNA repair pathways,

from yeast to mammalian cell lines, results in an increased

level of genomic instability (206, 207). While NHEJ is

required for V(D)J recombination, this process is a specific

example of the more general function of this pathway: to

appropriately resolve DSBs, regardless of the causative agent.

As a result, fibroblasts from NHEJ-deficient mice have an

increased level of IR-sensitivity, as well as elevated levels of

spontaneously occurring chromosomal aberrations (93, 99,

153, 208–211).

The types of karyotypic abnormalities observed in these cells

include broken chromosomes, broken chromatids, chromo-

some gaps, chromosome fusions, aneuploidy, and as well as

translocations. In particular, recurrent translocations are diag-

nostic of a number of human tumors (212), and may result in

the deletion of tumor suppressor genes, the deregulation or

amplification of protooncogenes, or the production of novel

fusion proteins, all of which can promote oncogenesis (213).

Therefore, by limiting such genome instability, NHEJ has a

role in suppressing tumor formation.

When coupled with the inactivation of cell-cycle check-

points, the absence of NHEJ can lead to tumor formation

(213). While mice doubly deficient in any of the six known

NHEJ factors and p53 (NHEJ/p53) appear relatively normal at

birth, these mice all have an increased mortality rate and an

elevated level of tumor incidence (98, 99, 153, 154, 214–

216). Nearly all tumors arising in NHEJ/p53-deficient mice

(with the notable exception of Artemis/p53 mice, see below)

are B220þIgM–CD43þ progenitor (pro)-B-cell lymphomas,

characterized by a marker der(12)t(12;15) translocation

(referred to as C12;15) (99, 153, 217–220) and co-

amplification of IgH and c-myc on a complex translocation

product, referred to as a complicon (220).

Based on the identification of several key structural inter-

mediates by fluorescence in situ hybridization and chromo-

some painting techniques, the molecular mechanisms

underlying the transformation of NHEJ/p53 pro-B cells have

been elucidated (217, 220) (Fig. 3). In the absence of p53,

Rooney et al � The NHEJ pathway in lymphocyte development

Immunological Reviews 200/2004 123

unrepaired RAG-induced DSBs at the IgH locus on chromosome

12 are allowed to persist into S phase, where the broken

chromosome 12 is replicated and recombines with chromosome

15 to produce a clonal C12;15, as well as a dicentric 12;15

chromosome. This dicentric intermediate is then subjected to

multiple rounds of a breakage–fusion–bridge (BFB) cycle,

terminated following recombination with the distal portion of

a third unrelated chromosome, thereby resulting in the forma-

tion of a complicon harboring co-amplified IgH and c-myc.

Despite the progress made in understanding the role of

NHEJ in suppressing lymphomas in mice, it remains unclear

what role defects in NHEJ play in human tumors. While there

is some evidence that polymorphisms of NHEJ factors, par-

ticularly ligase IV, can influence the susceptibility to various

cancers (147, 221–224), such analysis is complicated by the

fact that Artemis is the only NHEJ factor known to be inacti-

vated in humans, potentially due to a more severe impact of

NHEJ mutations in human versus mouse cells (225). In this

context, hypomorphic alleles of Artemis have been associated

with human lymphoid malignancies harboring chromosomal

translocations (226), although it is unclear whether this is

secondary to infection with Epstein–Barr virus.

Artemis/p53-deficient mice also develop pro-B cell tumors

at an age comparable to other NHEJ/p53 mice, and c-myc

amplification and C12;15 translocation were observed in a

subset of Artemis/p53 tumors (216). However, rather than

c-myc, a majority of Artemis/p53 pro-B cells exhibit amplifica-

tion of the related oncogene N-myc. As amplification of both

N-myc and c-myc appears to occur through similar BFB cycles

(216), it is of interest to speculate why only c-myc amplifica-

tion is observed in other NHEJ/p53 mice. While mutation of

either c-myc or N-myc is lethal embryonically (227, 228),

replacement of c-myc with N-myc has little effect on develop-

ment (229), suggesting that the functional difference between

the two lies in their expression patterns. Given the appearance

of both N-myc and c-myc amplification in Artemis/p53 tumors,

it is likely that the oncogenic potential of N-myc and c-myc is

also largely equivalent. Therefore, whether due to strain dif-

ferences or the distinct impact of the loss of different NHEJ

factors, the conserved amplification of c-myc in most NHEJ/

p53 pro-B-cell tumors appears to be the result of preferential

translocation of IgH into the regions surrounding c-myc, rather

than selection for c-myc.

Switch translocations

Aberrant repair of V(D)J-associated DSBs is not alone in its

ability to promote chromosomal translocations. Translocation

breakpoints involving IgH S regions and c-myc have been cloned

from a number of lymphomas from both mice and humans

(213), indicating that inappropriate resolution of CSR inter-

mediates can promote lymphomagenesis as well. Unlike the

complex translocations observed in NHEJ/p53 pro-B cells,

these translocations tend to be simple and balanced, with the

switch region translocated directly into either the first exon or

the first intron of c-myc. Whether NHEJ deficiencies are capable

of promoting switch-region translocation in mature B cells

remains to be determined. However, H2AXþ/Dp53–/– mice

develop a wide spectrum of lymphoid tumors, including

B-lineage tumors with translocation breakpoints involving

Sm and c-myc (202), suggesting that haploinsufficiency for

H2AX can promote CSR-associated translocations in a

p53-deficient background.

Concluding remarks

While much is understood about the NHEJ pathway, there is

clearly much left to be determined. Both genetic and biochem-

ical data suggest that V(D)J recombination requires additional

factors (75, 230, 231); these may include polynucleotide

kinase, which restores 50 phosphate ends (232), or one of

several gap-filling polymerases (233–236). Similarly, general

DSB repair by NHEJ may require other nucleases to deal with

ends that cannot be processed by Artemis, such as WRN (107,

136), FEN-1 (135), or the Mre11 complex (137).

One additional factor that modulates the resolution of V(D)J

intermediates is RAG itself. Paired cleavage of gene segments

ensures that RAG does not introduce a single DSB within the

genome (237). Furthermore, following the generation of two

DSBs, RAG remains bound in a post-synaptic cleavage com-

plex, holding DNA ends in proximity to one another (238). In

this manner, RAG appears to facilitate the productive inter-

action between cleaved gene segments. Similarly, it remains to

be determined how the RAG complex influences the choice of

DNA repair pathways and how the post-synaptic cleavage

complex interacts with various members of the NHEJ pathway.

Further examination of the potential role of DNA damage

response pathways in V(D)J recombination is warranted.

While H2AX or 53BP1 is not required for V(D)J recombin-

ation per se (239–242), H2AX may prevent aberrant processing

of V(D)J ends (201). Furthermore, in cells with multiple DSBs,

NHEJ tends to join ends from a given DSB back to each other,

rather than to those of another DSB (210). Therefore, these

ends must be juxtaposed with one another within the context

of chromosomal DNA. While this may be achieved in V(D)J

recombination by the RAG proteins, the anchoring of general

Rooney et al � The NHEJ pathway in lymphocyte development

124 Immunological Reviews 200/2004

DSBs may be promoted by g-H2AX and by its associated DNA

damage factors (201). Further elucidation of such potential

functions of these proteins may yield significant insights into

both lymphocyte-specific and more general translocations.

Unlike V(D)J recombination, little is understood about the

resolution of CSR. Clearly, AID and germline transcription are

required, but the intermediates generated by this process

remain ill-defined. Particularly, while most studies point to

the introduction of DSBs during CSR, direct evidence has

remained elusive. In the same context, the role of NHEJ in

CSR needs to be clarified. Examination of CSR in the absence of

XRCC4 or ligase IV should provide the most tractable system to

evaluate this question, as both factors are required for all NHEJ

reactions, and they do not appear to have functions outside of

this pathway. Similarly, it remains to be determined how the

productive interaction of two switch regions is ensured. H2AX

has been proposed to anchor DNA ends through multiple

protein–protein and protein–DNA interactions (201). There-

fore, systematic evaluation of the role of H2AX-associated

factors in CSR may give further insight into the joining phase

of this reaction.

A complete index of the function(s) of DNA-PK is also

required. This complex appears to have a multitude of roles,

not all of which are involved in NHEJ per se (101, 243), and

some of which, such as the protection of telomeres from

fusions (102), appear to be in direct contrast with its role in

NHEJ. Therefore, the function of the holoenzyme most likely

is modulated in some manner, possibly by interacting with

various partners in different contexts or by post-translational

modification.

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