Nonhomologous-end-joining repair pathway

In response to DSBs, cells have developed two general types of repair: homologous recombination (HR) and nonhomologous end joining (NHEJ). HR is generally limited to the late S phase and G2 phase of the cell cycle in mammalian cells, and uses information from a homologous template to accurately repair breaks when sister chromatids present readily available templates. NHEJ is the main DNA-repair pathway that mediates the joining of broken regions of DNA that lack extensive homology, and is the principle mechanism used in vertebrate cells during the G1 phase of the cell cycle. Seven mammalian factors have now been identified as crucial NHEJ components. The DNA-binding subunits Ku70 (XRCC6) and Ku80 (XRCC5) together with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs or PRKDC), form the DNA-PK holoenzyme, which is involved early in the recognition of DSBs. Activated DNA-PK holoenzyme recruits other NHEJ proteins including artemis (DCLRE1C), XRCC4, DNA ligase 4 (LIG4) as well as DNA polymerase µ (Polµ or POLM) to the site of DNA damage (Fig. 1). After phosphorylation by DNA-PKcs, the endonuclease artemis resolves complex DNA ends, such as the heterologous-loop and stem-loop DNA structures, which contain single-stranded DNA adjacent to double-stranded DNA. Polµ is associated with both the Ku heterodimer, and DNA ligase IV (LIG4) and might have a role in gap filling during NHEJ, but is not critical for ligation. LIG4, XRCC4 and cernunnos-XRCC4-like factor (XLF or NHEJ1) are also required for the ligation reaction that rejoins the DSBs.

Figure 1.

VDJ recombination. DNA is uncoiled at transcription factories within the cell, where the associated recombination and repair proteins colocalise. (a, b) The lymphoid-specific recombinase-activating gene 1 and 2 (RAG1 and RAG2) proteins recognise and bind the recombination signal sequences (RSS) that flank the VDJ gene segments, and introduce site-specific DNA double-strand breaks (DSBs) through their endonuclease action. (c) The phosphorylated blunt signal ends and the covalently sealed hairpin intermediate of the coding end are held together by the RAG complex. (d) The MRE11–RAD50–NBS1 (MRN) complex binds the broken DNA ends and activates the ATM (ataxia-telangiectasia mutated) protein, which initiates cell cycle arrest and attraction of the repair proteins γH2AX, 53BP1, RNF168 and others, to stabilise the damaged chromatin. (e) (Step 1) The Ku70–Ku80 heterodimer binds the coding ends and recruits DNA-PKcs and artemis, which is required to open the hairpin intermediates. The covalently sealed hairpin intermediate is randomly nicked by the DNA-Pkcs–Artemis complex, which generates a single stranded break with 3′ or 5′ overhangs. (Step 2) XRCC4, DNA ligase IV (LIG4) and cernunnos-XLF (C-XLF) coassociate and are recruited to the ends. The signal ends are directly ligated by the XRCC4–LIG4–C-XLF complex. The opened hairpin intermediate is modified by polymerases, exonucleases and the lymphoid-specific terminal deoxynucleotidyl transferase (TdT), before (Step 3) repair and ligation by the XRCC4–LIG4–C-XLF complex.

DNA-mismatch repair

Mismatch repair (MMR) corrects DNA mismatches generated during DNA replication, preventing mutations from becoming permanent in dividing cells. Nucleotide misincorporation generates DNA base–base mismatches during DNA synthesis at a variable rate, depending on many factors, including which specific DNA polymerases are present. The DNA MMR pathway is highly conserved, and specific primarily for base–base mismatches and insertion–deletion mispairs generated during DNA replication and recombination. A number of human MMR proteins have been identified based on homology to Escherichia coli MMR proteins. Human MSH2 forms a heterodimer with MSH6 or MSH3 to form MutSα or MutSβ respectively, both of which are ATPases with a crucial role in mismatch recognition and initiation of repair. MutSα preferentially recognises base-to-base mismatches and small nucleotide insertion–deletion mispairs of one or two nucleotides, whereas MutSβ preferentially recognises larger insertion–deletion mispairs. Four human MutL homologs (MLH1, MLH3, PMS1, PMS2) have been identified. MLH1 forms a heterodimer with PMS2, PMS1 or MLH3 to form MutLα, MutLβ or MutLγ, respectively. Mutlα is required for mismatch repair by resolving four-stranded DNA structures (DNA Holliday junctions), which form during HR in meiosis. Heterodimers of MSH5 and MSH4 are postulated to form a ‘sliding clamp’ on DNA, and serve as scaffolding for the recombination machinery, including the DNA-repair proteins MLH1 and PMS2. MutLγ has a role in meiosis, but no specific biological role has been identified for MutLβ. MutLα regulates termination of mismatch-provoked excision. Other enzymes involved in mismatch repair include proliferating cellular nuclear antigen (PCNA), DNA polymerase δ (Polδ or POLD1) and DNA ligase I (LIG1). PCNA interacts with MSH2 and MLH1 and is thought to have roles in the initiation and DNA-resynthesis steps of mismatch repair. PCNA also interacts with MSH6 and MSH3 and might help to localise MutSα and MutSβ to mispairs in newly replicated DNA. DNA resynthesis and ligation is performed by LIG1.

DNA-repair proteins and generation of lymphocyte antigen receptors

Effective immunity requires recognition of foreign antigens, requiring the generation of ≈10¹⁸ genetically diverse cells, each with a unique receptor that recognises a unique antigen–MHC combination. In jawed vertebrates, these receptors are created by breaking, randomly resorting and joining DNA sequences coding for the antigen-capture region of the receptor, by adapting the ubiquitous DNA-repair mechanisms that maintain genome stability. Recombination is a site-specific event that occurs at the T-cell receptor (TCR) α-, β-, γ- and δ-chain loci, and the B-cell receptor (BCR) immunoglobulin heavy (IgH), and immunoglobulin κ or λ light (IgL) chain loci. Recombination occurs between component variable (V), junction (J), and for TCRβ, TCRδ and BCR IgH loci, diversity (D) gene segments, with the fused VJ or VDJ coding sequence subsequently joined to a constant region segment through RNA splicing. Two recombination-activating gene proteins (RAG1 and RAG2) introduce site-specific DSBs at conserved noncoding DNA sequences adjacent to the points at which recombination occurs, known as recombination signal sequence (RSS) sites, either side of the segments to be rearranged, during the G1 phase of the cell cycle.

After the introduction of DSBs at the coding-sequence–RSS junction, two types of DNA ends arise: coding sequence ends that reconstitute the Ig and TCR genes are generated as hairpin intermediates, and noncoding signal ends containing the motifs targeting site-specific cleavage, which are generated as blunt double-stranded DNA ends. ATM is not required for cleavage of coding joint hairpin intermediates (Ref. Reference Riballo1), but helps to stabilise DNA ends in the RAG postsynaptic cleavage complex, facilitating NHEJ repair of VDJ-recombination-associated breaks (Refs Reference Bredemeyer2, Reference Huang3, Reference Helmink4). ATM, nibrin, γH2AX and 53BPI are associated with RAG-induced DSBs in developing lymphocytes and localise to DSBs and the chromatin region surrounding the recombining loci (Refs Reference Perkins5, Reference Chen6, Reference Celeste7, Reference Stracker8, Reference Difilippantonio9). ATM might contribute to the efficiency of VDJ recombination by activating cell-cycle-checkpoint proteins, which enables monitoring of recombination intermediates. In the absence of ATM, lymphocytes with RAG-induced DSBs might enter the S phase of the cell cycle, which leads to a reduction in productive VDJ recombination and an increased number of abnormal translocations involving Ig and TCR loci (Ref. Reference Kobayashi10).

DSBs are repaired using the NHEJ repair pathway (Table 1). The Ku70–Ku80 heterodimer binds DNA ends present at RAG1- or RAG2-generated coding ends and recruits DNA-PKcs, which phosphorylate and activate artemis endonuclease activity to process the coding sequence hairpin intermediates. Following cleavage, coding and signal ends are directly ligated by the XRCC4–LIG4–C-XLF complex (Ref. Reference Lieber11) (Fig. 1). Rejoining of signal ends does not require DNA-PKcs or artemis. Furthermore, VDJ recombination is not completely abolished if any of the seven NHEJ proteins are impaired, because an alternative end-joining pathway exists, in which the frequent use of microhomology and excessive deletions are characteristic. RAG proteins have an essential role in the joining phase of V(D)J recombination, but allow a small degree of alternative NHEJ activity (Ref. Reference Corneo12).

Table 1.

Proteins in the nonhomologous end-joining DSB-repair pathway

Abbreviations: LIG4, DNA ligase 4; Polλ, DNA polymerase λ; Polμ, DNA polymerase μ; TdT, terminal deoxynucleotidyl transferase.

T- and B-cell-receptor recombination occurs in the thymus and bone marrow, respectively. Early lymphocyte progenitors undergo successive stages of lineage commitment, generating a functional lymphocyte receptor repertoire. Between critical developmental stages of VDJ rearrangement of the T-cell β- and α-chain, and B-cell IgH and IgL chain, the lymphocyte precursors undergo intense proliferation. During this phase, cells experience the normal replicative stress of proliferating cells, and in doing so, accumulate abnormal replication intermediates, normally resolved by Bloom syndrome protein (BLM) (Refs Reference Babbe13, Reference Babbe14).

DNA-repair proteins and immunoglobulin class-switch recombination

Maturation of the antibody repertoire is required to optimise antibody responses with high antigen affinity. Antibody maturation occurs in the germinal centres of secondary lymphoid organs in response to antigen- and T-cell-driven activation; B cells proliferate vigorously, dividing every 6–8 h and accumulating abnormal replication intermediates acquired during replicative stress, which are resolved by Bloom syndrome protein. Class-switch recombination (CSR) is a somatic DNA-arrangement process, which results in a switch in the IgH constant region of the BCR, expressed from the region encoded by Cµ, to a downstream constant region such as that encoded by Cα, Cγ or Cη. Switch (S) regions lie in the J-C intron. DNA recombination occurs between Sµ and the S region upstream of the C region for the particular antibody isotype, resulting in deletion of the intervening DNA. This results in the production of antibodies of different isotypes (IgG, IgA and IgE) with the same V(D)J specificity and therefore the same antigen affinity (Ref. Reference Iwasato15). Activation-induced cytidine deaminase (AID), a B-cell-specific enzyme that is crucial for CSR, induces DSBs to initiate CSR (Refs Reference Muramatsu16, Reference Revy17). AID selectively deaminates cytosine residues to uracil in the switch and variable regions (Ref. Reference Bransteitter18). Uracil DNA glycosylase (UNG) removes uracil, producing an abasic site, which is cleaved by one of the base excision–repair enzymes to create a DNA single-strand break (SSB) (Refs Reference Rada19, Reference Guikema20). The MMR proteins MSH2–MSH6 recognise U at U:G mismatched bases, and create a further SSB (Ref. Reference Xue, Rada and Neuberger21). If a particular U is on the complementary strand to a previous SSB, a DSB results, enabling CSR to occur (Ref. Reference Wilson22) (Fig. 2). PMS2, acting as a heterodimer with MHL1, converts AID- and UNG-induced SSBs into the DSBs required for CSR by stabilising the recombination intermediate (Refs Reference Schrader, Vardo and Stavnezer23, Reference Péron24). MSH5 is also involved in CSR, and might have a specific role in facilitating CSR between Sμ and Sα (Ref. Reference Sekine25).

Figure 2.

Class-switch recombination between Cμ and Cγ₁. (a) Activation-induced cytidine deaminase (AID) selectively targets cytosine bases in the Cμ and Cγ₁ switch regions of the IgH constant region in the B cell receptor gene (Step 1). Cytosine residues are deaminated to uracil (Steps 2 and 3). (b) Uracil DNA glycosylase (UNG) removes uracil, producing an abasic site, which is cleaved by one of the base excision-repair enzymes (Step 1) to create a DNA single-strand break (Step 2). The mismatch-repair proteins MSH2–MSH6 also recognise U at U:G mismatched bases, and create a further DNA single-strand break, resulting in a DNA double-strand break (DSB) (Step 3). (c) DSB repair is achieved mainly through nonhomologous end joining. ATM (ataxia-telangiectasia mutated) is required for the repair and might recruit or activate 53BP1, γH2AX and RNF168, which are likely to be involved at the synapsis. (d) ATM might also phosphorylate artemis, which is required to repair a subset of DSBs, and organises the damaged DNA ends for subsequent repair steps, or arrests cell cycle progression until the repair is complete. Nibrin/NBS1, MRE11 and RAD50 are phosphorylated by ATM and might be required to activate ATM in CSR. MRE11, nibrin, Ku70–Ku80, DNA-PKcs, XRCC4 and DNA Ligase IV are likely to be involved in the final repair, as is cernunnos-XLF.

A significant role for Bloom syndrome protein is unlikely during CSR (Ref. Reference Babbe26), although it does interact with MLH1 and MSH6 (Refs Reference Schrader, Vardo and Stavnezer23, Reference Pedrazzi27, Reference Pedrazzi28). CSR DSB repair is achieved mainly through NHEJ during the G1 phase of the cell cycle (Ref. Reference Schrader29), although an alternative end-joining mechanism is used when there is impairment of NHEJ (Ref. Reference Yan30). ATM is required for efficient CSR, although the exact function remains unclear. ATM is also associated with MMR factors, including MSH2, MSH6, MLH1 and PMS2 (Ref. Reference Matsuoka31). Additionally, nibrin, MRE11, γH2AX and 53BP1, foci of which can be detected at the DSB switch region during CSR, are phosphorylated by ATM (Refs Reference Berkovich, Monnat and Kastan32, Reference Burma33, Reference Kobayashi34, Reference Ward35). One role of ATM might be to recruit or activate these factors, and organise the damaged DNA ends for subsequent repair steps, or arrest cell cycle progression until the repair is complete. ATM might have a more direct role in the end-processing step through phosphorylation of MRE11 or artemis – two proteins that participate in NHEJ. Artemis is downstream in the ATM signalling pathway for repair of a subset of radiation-induced DSBs, but is dispensable for ATM-dependent cell-cycle-checkpoint arrest (Ref. Reference Riballo1), and also appears dispensable for processing of DSBs for efficient CSR (Ref. Reference Rooney36), although it might be required for repair of the CSR-related chromosomal breaks at the Ig locus (Ref. Reference Franco37) and for the resolution of a subset of breaks generated during CSR (Ref. Reference Rivera-Munoz38). The altered pattern of CSR junctions in artemis-deficient patients also suggests that this protein is required in the predominant NHEJ pathway during CSR (Ref. Reference Du39). Deficiency in MRE11, nibrin, Ku70–Ku80, DNA-PKcs or LIG4 appears to alter the balance between the predominantly used NHEJ and alternative end-joining mechanism in CSR DSB repair, suggesting that these proteins are also involved directly or indirectly in CSR (Ref. Reference Pan-Hammarstrom40).

Little is known about the proteins or mechanism involved in the alternative end-joining mechanism. Poly (ADP-ribose) polymerase 1 (PARP1) is involved in many cellular responses, including base excision repair (BER) and possibly HR (Ref. Reference Helleday, Bryant and Schultz41). It might be involved in the alternative end-joining pathway, with XRCC1 and DNA ligase III (LIG3) (Refs Reference Audebert, Salles and Calsou42, Reference Wang43, Reference Robert, Dantzer and Reina-San-Martin44). LIG1 and LIG3 are required for microhomology-mediated end joining but it is unknown which of these enzymes is involved in alternative end-joining during CSR (Ref. Reference Liang45).

DNA-repair proteins and somatic hypermutation

Although CSR and somatic hypermutation (SHM) occur together in germinal centres, neither is a prerequisite for the other because IgM can be mutated in the absence of any such feature in IgG or IgA isotypes (Ref. Reference Kaartinen46). SHM introduces random mutations into the BCR variable region, resulting in minor conformational changes, which enables positive selection of B cells that carry a BCR with high antigen affinity (Ref. Reference Storb47). SHM is initiated by AID, by RNA editing of variable region C to U residues (Ref. Reference Shivarov48). The MMR proteins MSH2–MSH6 recognise AID-induced U/G residues, and recruit the exonuclease EXO1 and DNA polymerase η (POLH) leading to G:C to T:A transversions (Ref. Reference Schanz49). Whilst NHEJ is not used in SHM, the MRN complex is involved in DNA cleavage at AID-induced abasic sites during SHM (Ref. Reference Larson50), and nibrin has a role in regulating strand-biased repair (Ref. Reference Du39). Bloom syndrome protein does not seem to be involved in SHM (Ref. Reference Sack51).

DSB-initiation defects

DSB-damage-sensing and cell-cycle-arrest defects

Nijmegen breakage syndrome

Nijmegen breakage syndrome (NBS) is associated with a characteristic facial appearance: receding forehead, receding mandible and prominent midface (‘bird-like’ facies) (Ref. Reference Weemaes69). Additional features include epicanthic folds, large ears and sparse hair with microcephaly and mild mental retardation. Patients are prone to sinopulmonary infection and are susceptible to B-cell-lineage lymphomas. Cellular immunity is consistently impaired in NBS patients; lymphcytopaenia is common, with reduced proportions of CD3 and CD4 T cells (Ref. Reference Digweed and Sperling70). Agammaglobulinaemia is reported in about a third of NBS patients, whereas in others the humoral immune deficiency is more variable. Deficiencies of IgA or IgG4, alone or in combination, are common (Ref. Reference Gregorek71). About 10% of patients have normal immunoglobulins. The immunodeficiency might result from reduced fidelity of VDJ recombination, because nibrin is involved with ATM in inducing cell cycle arrest during this process (Ref. Reference Xu72). Frequency of VDJ recombination in NBS patients is normal, however, with normal IgH rearrangement. The deficiency of serum IgG and IgA with normal or raised IgM is probably due to impaired CSR (Refs Reference Kracker73, Reference Reina-San-Martin74).

Chromosomal inversions and translocations, particularly chromosome 7:14 translocations are characteristic of NBS (Fig. 3). NBS cells are sensitive to DNA-crosslinking agents such as mitomycin C and diepoxybutane (DEB), in addition to IR. Patients with certain NBS1 mutations have features similar to those seen in Fanconi's anaemia (Refs Reference Nakanishi75, Reference Gennery76), although immunodeficiency is more pronounced, presumably because of the role of nibrin in VDJ recombination and CSR.

Figure 3.

Karyotype from a patient with Nijmegen breakage syndrome. (a) Chromosome t(7;14) rearrangement (arrows). (b) chromosomal breakage following exposure to 50 centiGray ionising radiation (arrows). (c) Multiradial formation (arrows) after culture for 72 hours following exposure to mitomycin C at 0.32 µg/ml for 60 minutes. (d) Chromosome breakage (arrows) following lymphocyte culture with diepoxybutane (DEB) for 72 hours (Reproduced with permission from the Paediatric HSCT Unit, Newcastle General Hospital).

DSB-recognition defects

DSB-processing defects

DSB-resolution defects

Other uncharacterised disorders

A number of genetically undefined disorders, with phenotypic and cellular features characteristic of NBS (Refs Reference Berardinelli113, Reference Maraschio114, Reference Hiel115) or Omenn-like SCID (Ref. Reference Wiegant116), but with no mutations in candidate genes, have been described, implicating further DNA-repair genes in human primary immunodeficiency.

Class-switch recombination and somatic hypermutation defects

Three autosomal recessive hyper-IgM syndromes have been described; they are caused by defects in DNA-break and DNA-repair mechanisms, which lead to decreased or abolished isotype switching and impaired somatic hypermutation.

Undefined defects

Further, as yet undescribed, gene defects give rise to a clinical picture of hyper-IgM syndrome. Hyper-IgM type 4 has been described in 15 patients, with characteristic features of recurrent respiratory and gastrointestinal tract infection, lymphoid hyperplasia and autoimmune features (Ref. Reference Imai125). CSR was defective, but SHM was normal. AID and UNG mutations were excluded in all patients.

A further clinical entity consisting of increased radiosensitivity but normal checkpoint arrest and NHEJ, increased levels of microhomology across Sμ-Sα switch junctions and a skewed SHM toward transitions at G or C residues has been described in five patients (Ref. Reference Péron126). All had recurrent respiratory infections; lymphoid hyperplasia and autoimmunity were also described. Raised IgM and decreased IgA and IgG levels were noted. A genetic defect has yet to be identified. A further group of patients has been described, in whom recurrent bacterial infection, autoimmunity and lymphadenopathy are observed, although the lymphadenopathy was less marked than in AID-deficient patients. There was a lack of class-switched B cells in these patients, although SHM was normal. There was no sensitivity to ionising radiation (Ref. Reference Durandy127). Defective CSR associated with growth hormone deficiency has been described in two patients in whom the molecular defect has yet to be discovered (Ref. Reference Kashef128, Reference Ohzeki129).

DNA ligase I

To date, one patient has been described with two compound missense mutations in LIG1 (Refs Reference Webster130, Reference Barnes131). Clinical features, overlapping with those of Bloom syndrome and ataxia telangiectasia, include intrauterine and postnatal growth retardation, developmental delay with normal cognitive development, dysmorphism with elf-like features, and photosensitivity. Immunodeficiency manifested as recurrent middle ear and respiratory infections from age 2 years, with evolving IgA deficiency, relative hypogammaglobulinaemia of IgG and normal IgM. There was an evolving lymphocytopaenia with poor proliferative response to mitogens. During teenage years, the respiratory status deteriorated and secondary sexual characteristics did not develop. At the age of 17 years, patches of cutaneous venous dilatation appeared mainly on the limbs, and there was some bulbar conjunctival telangiectasia. Hepatosplenomegaly developed with associated neutropaenia and increasing lymphopaenia. A liver biopsy showed lymphocyte infiltration of the portal tract, suggesting lymphoma. The patient developed a severe cutaneous herpes zoster infection and died from pneumonia at the age of 19 years. No information on end-joining of VDJ substrates, or on the use of microhomology, was available. An increase in the number of SSBs and DSBs in newly replicated DNA molecules was seen in an immortalised fibroblast line, possibly because of the failure of dealing with damage at replication forks (Ref. Reference Soza132). The cause of the immunodeficiency can only be conjectured, given the paucity of data. There is no evidence linking LIG1 with VDJ recombination (Ref. Reference Petrini133). LIG1 forms a complex with nibrin, and both colocalise at replication factories to repair DSBs by homologous recombination at stalled replication forks (Ref. Reference Vago134), suggesting that defects in LIG1 are associated with failure to repair DNA damage during lymphocyte proliferation, rather than failure to complete NHEJ in TCR and BCR formation. The finding of low IgA and IgG, but normal IgM is tantalising, and further work needs to be done to investigate what role, if any, LIG1 has in CSR.

Bloom syndrome

Bloom syndrome is an autosomal recessive disorder characterised by proportionate pre- and postnatal growth deficiency, photosensitive, telangiectatic, hypo- and hyperpigmented skin, predisposition to malignancy and chromosomal instability. There is an increased incidence of diabetes mellitus. Immunodeficiency, although common, is variable and generally not severe (Refs Reference Hütteroth, Litwin and German135, Reference Van Kerckhove136), although life-threatening infection can occur (Ref. Reference German137). Low concentrations of one or more immunoglobulin isotypes are most often found (Refs Reference Hütteroth, Litwin and German135, Reference Van Kerckhove136, Reference Kondo138). Impaired T cell proliferation, diminished CD4⁺ T-cell numbers and impaired function have been described in Bloom syndrome patients (Refs Reference Hütteroth, Litwin and German135, Reference Taniguchi139). There is a characteristic increase in sister-chromatid exchange seen upon cytogenetic analysis (Fig. 4). Bloom syndrome protein has no role in VDJ recombination (Refs. Reference Babbe13, Reference Hsieh, Arlett and Lieber140), and has only a minor role, if any, in CSR, although microhomology-mediated end joining was observed at Sμ-Sγ3 switch regions, possibly implicating BLM in the resolution phase of CSR (Ref. Reference Babbe14).

Figure 4.

Karyotype from a patient with Bloom syndrome. A symmetrical quadridradial (a, arrow), and a large increase in the number of sister chromatid exchanges (b, arrows) are visible. (Reproduced with permission from the Paediatric HSCT Unit, Newcastle General Hospital).

Fanconi anaemia

Fanconi anaemia is a clinically heterogenous autosomal recessive or X-linked disorder characterised by bone marrow failure, skeletal, renal, cardiac and gastrointestinal defects, hypopigmentation and predisposal to malignancy (Ref. Reference Alter141). The majority of immunological problems relate to bone marrow failure, but a few patients present early with significant or prolonged infections, more consistent with immunodeficiency (Ref. Reference Nakanishi75). Although cells from patients show hypersensitivity to agents causing DNA interstrand crosslinks, a few also demonstrate sensitivity to ionising radiation (Ref. Reference Mohseni-Meybodi, Mozdarani and Vosough142). Thirteen genes associated with Fanconi anaemia have been identified to date. Whilst most of the proteins form a core ubiquitin ligase complex, FAND2 is ubquitinated by this complex, and then colocalises to chromatin with other DNA-repair proteins, including the MRN complex (Ref. Reference Nakanishi75). The Fanconi anaemia proteins do not appear to have direct a role in lymphocyte receptor development or modification, and effects on immunity are more likely to be a result of interstrand crosslinks occurring during cellular development, resulting in bone marrow failure.