Key words: Double strand breaks; Non homologous end-joining; Chromosomal abnormalities; Human leukemias; Genomic instability; Repair; Predisposition; Fanconi anaemia; Bloom syndrome

Abstract

DNA double strand breaks (DSB) are considered the most lethal form of DNA damage for eukaryotic cells. DSB can either be properly repaired, restoring genomic integrity, or misrepaired resulting in drastic consequences, such as cell death, genomic instability, and cancer. It is well established that exposure to DSB-inducing agents is associated with chromosomal abnormalities and leukemogenesis. The non homologous end-joining (NHEJ) pathway is considered a major route for the repair DSB in mammalian cells. Although the mechanism(s) by which repair of DSB lead to leukemia are poorly understood, recent evidence is beginning to emerge that a poorly defined and error-prone branch of the NHEJ pathway plays a pivotal role in this process. This review discusses some of the ways in which error-prone NHEJ repair may be involved in the development of genomic instability and leukemia.

INTRODUCTION

DNA DSB can arise through exogenous treatment with agents, such as ionizing radiation and topoisomerase inhibitors, or can occur endogenously from such agents as reactive oxygen species (1, 2, 3, 4). DSB can also occur when replication forks encounter DNA lesions, such as ss DNA nicks or DNA cross-links (5, 6). Several lines of evidence suggest that the incidence of leukemia in mouse and man is associated with exposure to chemo- or radio-therapeutic agents, that cause DSB (7, 8). In order to maintain genomic integrity, higher eukaryotes have evolved multiple pathways for the repair of DSB; these include, homologous recombination (HR), non NHEJ and single strand annealing (SSA) (6). HR is a process in which a homologous DNA strand is used as a template for repair of DSB, and is essentially error-free (6). NHEJ joins together double stranded DNA ends after they are modified, and SSA occurs through excision of damaged bases in a single DNA strand and filling in the missing nucleotides; these two mechanisms, SSA and NHEJ, are essentially error-prone (6). NHEJ is regarded as the dominant mechanism for DSB repair in vertebrates, especially in G0 and G1 phases of the cell cycle (6,9). It is also known that NHEJ is essential for the repair of DSB made during the V(D)J recombination processes (10).

It is well established that leukemias that arose in atomic bomb survivors and (radio- and chemo-) therapy–related leukemias exhibit high levels of recurring chromosome abnormalities (11, 12, 13). Non random chromosomal abnormalities, such as translocations and deletions are the hallmark of human leukemias (14). These alterations disrupt the function of genes that contribute to leukemia initiation and progression. Several reports implicate DSB and their repair by NHEJ in the generation of some key chromosomal translocations in both childhood and adult acute myeloid and lymphoid leukemias (15). DSB-inducing agents, such as, ionizing radiation and topoisomerase inhibitors have demonstrated specific DNA cleavage in genomic regions of genes frequently involved in chromosomal translocations (16, 17, 18, 21). Moreover, sequence analysis of the translocation breakpoint junctions in some of these leukemias have revealed the presence DNA sequence homologies (up to 6bp), suggesting that NHEJ was responsible for religation of DSB at this locus (19, 20, 21).

Despite the sequence signatures of NHEJ repair at the breakpoint junctions of gene fusions in leukemias, little is understood of the mechanisms by which these chromosomal abnormalities are generated. In fact, previous reports have been confusing in this regard because either decreased or increased NHEJ activity have been reported to mediate chromosomal instability. For example, studies of mouse cells null for components of the NHEJ pathway suggest the importance of these proteins for protecting against genetic instability and tumorigenesis (22). On the other hand, several lines of evidence suggest that cells with an intact NHEJ pathway can give rise to chromosomal rearrangements in response to induction of high frequencies of DSB (23, 24, 25, 26). Furthermore, increased end-ligation efficiency and accompanying misrepair, have been demonstrated in sporadic myeloid leukemias (27). In addition, studies of NHEJ activity in some of the inherited chromosomal instability syndromes that predispose to leukemia suggest that error-prone NHEJ may be induced by different routes (28, 29, 30).

Non-homologous end joining repair of double strand breaks

NHEJ has been extensively characterized in rodent cells (31), identifying a pathway where the sub-unit proteins of the Ku70/Ku86 heterodimer bind free DNA ends at the sites of DSB with the subsequent recruitment of DNA-protein kinase (DNA-PKcs) to the sites of damage (Fig 1). Ku70/86 heterodimer bound to DNA-PK (the active form of DNA-PKcs) phosphorylates the bound Ku70/86 heterodimer (32, 33, 34). Because most breaks that result from oxidation or irradiation cannot be rejoined due to chemical damage, additional proteins are required to remove the damage and fill any gaps that are remaining (12). The recent elucidation of the structure of the Ku heterodimer suggests how it can control access to DNA ends and allow their limited processing (35). The Rad50-Mre11-Nbs1 complex, which contains helicase and exonuclease activities, may function in NHEJ by processing DNA ends (6). Once processed, targeted free ends are ligated by DNA ligase IV in conjunction with XRCC4 (36). Although end-joining reactions can be error-free, in normal cells a small minority of DSB can be misrepaired (6). One mechanism for misrepair involves joining of DNA ends at short stretches of sequence homology of up to 7 bases, and the subsequent loss of intervening sequences resulting in small deletions (≤ 20bp) (37, 38). Whether, misrepaired DSB result from use of a minor or default NHEJ pathway that is DNA-PKcs independent (see below), or whether the NHEJ pathway is inherently error-prone under varying circumstances, is unclear. In addition, proteins that participate in error-prone DSB repair remain to be elucidated.

DSB and NHEJ: Signatures in human leukemia
DSB and leukemia

Several lines of evidence suggest that exposure to DSB-inducing agents are strongly associated with the incidence of both childhood and adult leukemias (39, 7). An unfortunate consequence of the treatment of patients with cancer, particularly those receiving radiation and/or various types of chemotherapy, is that some patients develop secondary leukemias, usually acute myeloid leukemia (t-AML) (7). Furthermore, it is now well established that leukemias arising in atomic bomb survivors and radio (r)- and chemo (t)-therapy –related leukemias exhibit high levels of chromosome abnormalities in addition to the characteristic 5q-/monosomy 7 observed in most r-AML and t-AML (9, 10). Cancer patients treated with chemotherapeutic agents that inhibit topisomerase II (topo II), such as, etoposide (VP16) and Doxorubicin (Dox), are also prone to the development of therapy-related acute myeloid leukemia and acute lymphoid leukemia (t-AML, t-ALL) (40, 41, 42). Topo II is an enzyme that cleaves and disentangles supercoiled DNA during such activities as DNA replication. Drugs targeting topo II were found not to inhibit DNA cleavage, but to prevent religation of DSB. Thus, treatment with topo II inhibitors result in increased DSB and, presumably, this activity is related to the cytotoxic effects of the drug (41, 42). Despite the fact that these agents are therapeutically efficacious, treatment with these topo II inhibitors unexpectedly led to an increase in secondary leukemias. Several reports now demonstrate that topo II inhibitors induce DSB at specific cleavage sites within the MLL gene locus, frequently disrupted by chromosomal translocations at 11q23 (16, 17, 21, 43). These cleavage sites also correspond with DNAase 1 hypersensitive sites, suggesting that DSB have a preponderence for regions of chromatin in the “open and accessible” configuration (43). Furthermore, Betti et al. (2001) demonstrated that irradiation and a nongenotoxic activator of apoptosis, the anti-CD95 antibody, are each able to initiate translocations at the MLL exon 12 cleavage site. Moreover, analysis of the translocation junctions showed that they contained regions of microhomology consistent with their creation through NHEJ repair processes (21). These authors obtained further evidence for the involvement of NHEJ processes by showing that inhibition of DNA-PKcs compromised DNA end-joining, and eliminated MLL-restricted translocations (21). Approximately 80% of infants (<1 year of age) with AML or ALL have chromosome translocations involving chromosome 11q23 (44). It is widely accepted that infant leukemia develops in utero, based on the diagnosis of leukemia in newborns, and upon finding leukemia with identical rearrangements of the MLL gene in monozygotic twins (44, 45, 46). Proposed causative factors have included maternal exposure to agents that induce DSB, some of which inhibit topo II in vitro, and may have similar action to the above mentioned therapeutic drugs (44, 45, 46). Strick et al. (2000) determined that certain dietary bioflavanoids induce MLL break point cluster region (BCR) cleavage by inhibiting topo II, and, by inference, maternal ingestion of these agents could be associated with the induction of infant leukemia (16). Thus, strong evidence exists to support the notion that site–specific DNA cleavage represents the initial molecular event leading to chromosomal translocations and leukemia.
Microhomologous sequences: NHEJ signatures at translocation breakpoints
Evidence for the involvement of NHEJ in generating chromosomal translocations in human leukemias comes from the sequencing of breakpoint junctions in cells from infant, childhood and adult acute lymphoid leukemia (ALL) and AML patients (19, 20, 21, 47, 48). Wiemels and Greaves (20), among others, used an inverse PCR strategy to analyze the DNA sequences and structure surrounding the t(12;21) translocation breakpoint in childhood ALL. Specifically, these authors examined the TEL-AML1 gene fusion resulting from this translocation in nine cases. Breakpoints were scattered within 14 kb of intronic sequences between exons 5 and 6 of TEL and within 2 putative cluster regions within AML1 intron 1, and exhibited no characteristic signal sequences of the V(D)J recombinase, topo II consensus sites, or other sequence motifs associated with recombination. In contrast, junction regions revealed microhomologous sequences, small deletions and duplications characteristic of repair by NHEJ. Yoshida et al. (19) also examined the joining sequences of translocations involving the PML gene in denovo acute promyelocytic leukemia (APL) in 120 patients and 5 secondary APL patients. Again, although no clustering of breakpoints was demonstrated within the introns and no consensus sequence motifs were identified, the majority of breakpoint junctions were characterized by stretches of up to 7 nucleotides of microhomologous sequences, indicative of NHEJ repair (19). It is clear from the above discussion that NHEJ activity is strongly implicated in some of the chromosomal translocations in human leukemia. However, it is not known whether recurring chromosomal deletions arise through induction of DSB and repair by NHEJ. Detailed analysis of sequences at deletion breakpoint junctions found in therapy-related myelodysplastic syndromes and t-AML, for example, would be important to perform (49). Thus, despite microhomologous sequence signatures at translocation breakpoints, the actual mechanism(s) by which NHEJ generates chromosomal abnormalities is still poorly understood.

Increased NHEJ activity and repair infidelity in human leukemia

Several lines of evidence suggest that cells that are wild type for NHEJ pathways can undergo genomic rearrangments, in response to high dose ionizing radiation, albeit at very low levels (24, 25, 26). Furthermore, the frequency of genomic rearrangements in these cells was found to be dependent on the frequency of DSB induced and the position of the DSB in the genome. Rothkamm et al. (2001) demonstrated that genomic rearrangements occurred more frequently, for example, in highly repetitive regions of DNA (50). In fact, these authors demonstrated that the frequency of genomic rearrangements was drastically reduced in cells defective for components of the NHEJ pathway (24, 50). Gaymes et al. (2002) tested NHEJ activity in sporadic myeloid leukemias (27). Using established in vitro assays for DSB end-ligation efficiency and repair fidelity, nuclear extracts prepared from both cultured, primary acute, and chronic myeloid leukemia cells showed a significant (4-7-fold) increase in end-ligation efficiency and concomitant misrepair, characterized by large plasmid deletions, as compared with normal IL-2 stimulated peripheral blood lymphocytes and CD34+ myeloid progenitors (27). Strikingly, the NHEJ proteins, Ku70 and 86, and not DNA-PKcs, appeared responsible for the deletions in myeloid leukemias, as assessed by pre-incubating nuclear extracts with antisera against the key NHEJ proteins (Ku70, Ku86, and DNA-PKcs) and their blocking peptide controls (27). Sequence analysis of misrepaired plasmids revealed that illegitimate alignment of distant sequences of microhomology most likely resulted in ligation and deletion of intervening sequences (Fig 2). One explanation for this constutively increased NHEJ activity is that myeloid leukemias generate increased levels of endogenous DSB to which NHEJ pathways respond with “upregulated” activity.

Deficient NHEJ activity and repair infidelity in human leukemia
Mice null for key components of the NHEJ pathway are characterized by multiple defects, including increased incidence of lymphoma (22, 51). Inactivation of Ku70 and 86 is associated with growth retardation, hypersensitivity to ionizing radiation, severe combined immune deficiency (Scid), due to severly impaired V(D)J recombination, and chromosomal instability (6). These results suggest a mechanistic link between irradiation-induced DNA damage and its repair, particularly in relation to DSB formed during differentiation of B and T lymphocytes. No similar case of human immunodeficiency has so far been reported, suggesting that defects in NHEJ are rare, or cause embryonic lethality. However, Riballo et al. (1999) described for the first time a cell line (180BR) derived from an acute lymphoblastic leukemia patient who was highly sensitive to radiotherapy, with high frequencies of DSB, and an inactivating mutation in the NHEJ protein, DNA ligase IV (52, 53). They noted that although this defect resulted in pronounced radiosensitivity, DNA ligase IV deficiency was compatible with normal human viability, but may have conferred a predisposition to leukemia. Wang et al. (2001) subsequently demonstrated that while most vertebrate cells process DSB mainly using a fast, DNA-PKcs-dependent pathway (D-NHEJ), this pathway is inactivated in 180BR cells. Instead, DSB in 180BR cells are repaired using a slower NHEJ pathway that is independent of DNA-PKcs, and is error-prone (B-NHEJ) (54). Thus, in rare cases of sporadic leukemia, when DNA-PK-dependent NHEJ is inactivated, error-prone NHEJ may take over the repair of DSB, leading to chromosome instability and leukemia.

ONE OR MORE TSG LOCI IN 1p

Several observations suggest the existence of more than one commonly affected 1p locus in neuroblastoma. Patients with large 1p deletions have poorer outcome than patients with short or interstitial deletions[61]. While tumors with large 1p deletions were associated with adverse prognostic factors, such as diploidy or tetraploidy, and amplified MYCN, all tumors with small interstitial deletions were in the triploid range with a high proportion of tumors detected by mass screening. The existence of two distinct deleted regions was also suggested by LOH at polymorphic loci in clinically identified neuroblastomas [62, 63]. It appears that distinct loci are involved in neuroblastoma with and without MYCN amplification, since these tumors show different types of SRO[64]. Most MYCN-amplified neuroblastomas also have deletions of 1p, whereas MYCN single copy tumors show 1p LOH in only 15-20 percent of cases[42, 65]. 1p deletions of MYCN-amplified tumors are very large, always at least including a region from 1p35-1p36.1 to telomere. In contrast, in MYCN single copy cases, 1p deletions were described to be consistently smaller, and a commonly deleted region maps to 1p36.3. Thus, a second tumor suppressor locus inactivated by the 1p deletions in MYCN-amplified neuroblastomas has been postulated [63, 66]. This TSG was suggested to be localized at 1p35-36.1, just distal to the deletion border of the smallest 1p deletion found in MYCN-amplified cases[63, 67]. The smallest SRO of the MYCN single copy tumors is included into the larger SRO of MYCN-amplified tumors, implying that a distal suppressor locus in 1p36.2-3 must also be deleted in MYCN-amplified tumors.

Lessons learned about the NHEJ pathways from studies of leukemia predisposition syndromes

Several of the chromosomal instability syndromes which predispose to leukemias, have inactivated key genes in the HR pathway. This suggests that repair by error-free HR is defective in these cells. Repair of DSB in two such examples is discussed below:
Fanconi naemia
Patients with the inherited chromosomal instability syndrome, Fanconi anaemia (FA), are predisposed primarily to myeloid leukemias and preleukemias (55). Although the reason for the preponderance of myeloid malignancies in FA patients is unclear, the syndrome results from inactivation of one of many FA genes functioning in 8 different complementation groups, some of which are known to act in the HR pathway (56). Cells from FA patients are characterized by a spontaneous level of chromosomal breaks, rearrangements and deletions induced by DNA cross-linking agents (57). Smith et al. (58) demonstrated that lack of functional FA genes leads to a marked increase in infidelity of DSB repair by NHEJ. Using extrachromosomal recombination substrates, these authors examined V(D)J coding and signal joint formation in cells from patients with FA complementation groups C and D. They demonstrated a several fold increase in the frequency of aberrant rearrangements, consistent with excessive degradation of DNA ends generated during the V(D)J reaction (58). Although Lundberg et al. (29) found that end-joining events in nuclear extracts from FA A, D and C cells also demonstrated increased repair infidelity, this activity was found not to be dependent on NHEJ proteins, DNA-PKcs, Ku70, Ku86, DNA ligase IV, and Xrcc4. Furthermore, this DNA-PKcs-independent activity was also found to be reduced by 3-9-fold in FA patients (28, 29). Thus, although the mechanism is unclear, defective HR repair of DSB may lead to activation of the error-prone NHEJ in FA cells. The increased frequency of misrepair and illegitimate joining of DSB may then result in genomic instability and myeloid malignancies. Bloom syndrome
In contrast to patients with FA, those with the chromosomal instability disorder, Bloom syndrome (BS), are predisposed not only to myeloid and lymphoid leukemias but also to a spectrum of cancers seen in the general population (59). This disorder results from inactivation of the BLM gene (60)., important in the HR pathway. Although the actual mechanism remains unclear, there is evidence that BLM, a RecQ family helicase, is involved in repair of DNA damage during replication. One of the defining features of cells from BS individuals is chromosomal instability, characterized by elevated sister chromatid exchanges (SCEs), as well as chromosomal breaks, deletions, and rearrangements (61, 62). In studies from our lab, we tested whether defective HR in Bloom’s Syndrome (BS) cells may lead to compensatory increases in NHEJ activity. We found that BS cells have an increased NHEJ activity and accompanying repair infidelity, characterized by a high frequency of large plasmid deletions (≤400bp) in in vitro assays (30). Sequencing of these large deletions suggested that DNA ligation occurred through illegitimate rejoining of distant sites of micro-homology. Furthermore, we showed that the frequency of errors, including large plasmid deletions was dependent upon the presence of the Ku70/86 heterodimer, and not DNA-PKcs. The DSB misrepair frequencies were reduced to normal levels in the BS cells corrected following transfection of the full length BLM gene (30). Recently, Langland et al. (2002) have also reported increased misrepair in Bloom’s cells, but failed to find a high frequency of ligations at regions of microhomology in examination of small plasmid deletions from in vitro assays (63). Although the explanation for the differences in these studies is not clear, it does appear from both investigations that the defective HR in BS cells may lead to repair of DSB by the error-prone NHEJ pathway, resulting in increased genomic instability and predisposition to malignancy.

Summary (or error-prone NHEJ pathways to leukemia)

Much of the data in sporadic, therapy-related leukemia, and the leukemia predisposition syndromes suggest that error-prone pathways for the repair of DSB, most likely those from the NHEJ pathway, may be critically involved in the generation of chromosomal instability and leukemia (Fig. 3). This pathway is likely to permit illegitimate alignment of regions of microhomology surrounding DNA ends (Fig 3). However, the signaling molecule(s) that trigger activation of this error-prone pathway are unknown at present, and its components remain to be elucidated. Thus, the activation of error-prone NHEJ should be regarded as yet another pathway leading to a process of genomic instability in human malignancies. In turn, the specific alterations noted in neoplastic cells are probably the result of selection within this process for those rare changes that give cells an advantage during the course of tumor progression.

MYCN AMPLIFICATION

Amplified MYCN is one of the most prominent genomic abnormalities of neuroblastomas, and is prototypic for the significance of proto-oncogene amplification in tumorigenesis. It was originally detected by expression profiling of oncogenes in human neuroblastoma cells [76]. Because cytogenetically analyzed neuroblastoma cells contained conspicuous chromosomal abnormalities, homogeneously staining regions (HSRs) or double minutes (DMs) indicative of amplified DNA, it was suspected that the high expression seen in expression profiles was the consequence of gene amplification. This suspicion was verified by DNA analyses[76]. These original studies also established the amplification of MYCN in a neuroblastoma tumor in addition to cell lines. A subsequent study confirmed amplified MYCN in a substantial proportion of neuroblastomas [77].
DMs which predominante in primary tumors, and HSRs in cell lines were found as the chromosomal sites of amplified MYCN[78]. HSRs are generally located on different chromosomes, not at the resident site 2p24 of MYCN [78, 79]. Amplification values in neuroblastoma may range between 5-fold and more than 500-fold, usually values of around 50- to 100 fold are seen in tumors. Neuroblastoma cells lacking amplification are not necessarily single copy for MYCN. Instead, MYCN can be duplicated at 2p24, as shown by fluorescence in situ hybridization[80]. In addition to cell lines, duplication has been seen in primary tumors using FISH and CGH[22]. No systematic survey of tumors has yet been carried out, and therefore the frequency of duplications among tumors has remained unknown. The biological significance of duplication is emphasized by reports of neuroblastomas arising in children with constitutional duplication of 2p (p23-pter) including the MYCN locus[81, 82]. It is unclear whether duplication represents a prelude to amplification or an alternative pathway for activating the oncogenic potential of MYCN. MYCN copy number is usually consistent within a tumor, not only at different tumor sites, but also at different times in vivo[83]. This suggests that amplified MYCN, in positive tumors, is generally present at the time of diagnosis. FISH analyses of primary tumors, which allow detection at single cell level, have revealed that individual cells from MYCN amplified tumors typically stray widely from the mean copy numbers suggested by molecular analyses[84, 85]. The molecular pathways by which MYCN is amplified have remained enigmatic. The end-point of amplification appears to be a tandem arrangement of unit size amplicons in an HSR on a chromosomal position different form 2p24 with retention of the single copy MYCN at 2p24. These observations have suggested a model of the amplification process involving unscheduled replication and recombination to produce circular extrachromosomal elements, which could be visualized as a DM. At some point this extrachromosomal DNA could integrate into any chromosomal site and undergo several cycles of in situ amplification.

The complexity of amplified DNA encompassing MYCN can range from 100kb to more than 1 Mb [86]. A core 100- to 200 kb domain encompassing MYCN has been found consistently without rearrangements. In most HSRs several hundred kilobase DNA segments of unit size length without noticeable rearrangement compared with the normal genomic organization are present in an direct repeat head-to-tail tandem arrangement[86]. The large size of the amplified DNA in relation to the size of the MYCN gene raises the possibility that additional genes are coamplified. From several technological strategies to identify coamplified genes MYCN has emerged as the only consistently amplified gene, although in about 50-70% of the MYCN-positive cases the DDX1 gene, which maps within 400 kb 5’ of MYCN is found coamplified[87, 88]. More recently, neuroblastoma amplified gene (NAG) was also shown to be coamplified in 70% of MYCN- amplified neuroblastomas and to map telomeric to MYCN[89]. So far, no amplification of DDX1 or NAG without concomitant MYCN amplification has been noted, suggesting that MYCN is functionally responsible for the maintenance of the 2p24-amplified DNA.
These observations have raised the question, whether additional genes may be amplified in neuroblastoma, which are not restricted to the MYCN amplicon. In neuroblastoma cell lines, additional DMs or HSRS were identified not harboring MYCN. Using reverse genomic hybridization, amplified DNA was found to be derived from chromosome 12 band q13-14. Subsequent analyses showed 30-40-fold amplification of the MDM2 gene, abundantly expressed, both in some cell lines and a primary tumor, in addition to amplified MYCN[90]. This non-syntenic amplification of the MDM2 gene appears to be a rare event in neuroblastoma cells and has been seen exclusively in conjunction with amplified MYCN. More recently, co-amplification of the homeobox gene, MEIS1, which map to band 2p14, was described in the neuroblastoma cell line IMR-32[91]. Again, amplified MEIS1 was exclusively observed together with MYCN amplification. It is possible that neuroblastoma cells with several amplified genes have a particularly pronounced genomic instability, although it is not clear why amplification in neuroblastoma cells is restricted to certain genetic loci.

Acknowledgements

FVR is funded by the elimination of leukemia Fund (ELF). I would like to thank the following individuals for critical reading of this review: Professor Ghulam Mufti and Dr Shaun Thomas, GKT School of Medicine (London, UK), Professor Ian Hickson,Weatherall Institute of Molecular Medicine (Oxford, UK), Drs. Pamela Strissel and Reiner Strick, Universität of Erlangen-Nürnberg (Erlangen, Germany), and Professor Stephen Baylin, Johns Hopkins School of Medicine (Baltimore, USA).

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Figure 1. Repair of a double strand break by NHEJ. Ku heterodimers bind free DNA ends and recruit DNA-PKcs. DNA ends are unwound and processed possibly by the Rad50-MRE11-Nbs1 complex. Processed DNA ends are subsequently ligated by the DNA ligase IV/ XRCCIV complex.

Figure 2. Puc18 plasmid DNA sequences showing Eco RI restriction site (arrows) and positions of microhomologous DNA sequences (underlined). Deletions occur through illegitimate pairing of distant regions of microhomology followed by ligation and excision of intervening DNA sequences.

Figure 3. Model for the creation of DNA deletions and translocations by error-prone NHEJ apparatus in vivo. Error-prone NHEJ repair pathways may be activated in the following ways: High frequencies of DSB (e.g. high dose radiation); Inactivation of key genes involved in HR repair (e.g. leukemia predisposition syndromes), Inactivation of DNA-PK-dependent NHEJ repair (180BR); Genetic mutations in sporadic leukemias? Error-prone repair can then illegitimately ligate DSB, leading to possible chromosomal abnormalities.

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