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Evidence for replicative repair of DNA double-strand breaks leading to oncogenic translocation and gene amplification.

Difilippantonio MJ, Petersen S, Chen HT, Johnson R, Jasin M, Kanaar R, Ried T, Nussenzweig A - J. Exp. Med. (2002)

Bottom Line: Subsequent DNA repair events juxtaposing IgH and c-myc are mediated by a break-induced replication pathway.Cycles of breakage-fusion-bridge result in amplification of IgH/c-myc while chromosome stabilization occurs through telomere capture.Thus, mice deficient in NHEJ provide excellent models to study the etiology of unbalanced translocations and amplification events during tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. difilipm@mail.nih.gov

ABSTRACT
Nonreciprocal translocations and gene amplifications are commonly found in human tumors. Although little is known about the mechanisms leading to such aberrations, tissue culture models predict that they can arise from DNA breakage, followed by cycles of chromatid fusion, asymmetric mitotic breakage, and replication. Mice deficient in both a nonhomologous end joining (NHEJ) DNA repair protein and the p53 tumor suppressor develop lymphomas at an early age harboring amplification of an IgH/c-myc fusion. Here we report that these chromosomal rearrangements are initiated by a recombination activating gene (RAG)-induced DNA cleavage. Subsequent DNA repair events juxtaposing IgH and c-myc are mediated by a break-induced replication pathway. Cycles of breakage-fusion-bridge result in amplification of IgH/c-myc while chromosome stabilization occurs through telomere capture. Thus, mice deficient in NHEJ provide excellent models to study the etiology of unbalanced translocations and amplification events during tumorigenesis.

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Proposed model for rearrangement events in the Kuβˆ’/βˆ’p53βˆ’/βˆ’ and Kuβˆ’/βˆ’p53βˆ’/βˆ’ Rad54βˆ’/βˆ’ pro-B cell lymphomas. RAG proteins initiate DNA DSB at the IgH D and J gene segments (step 1). Due to the absence of the Ku holoenzyme, the DSB cannot be efficiently and correctly repaired. The IgH DSB attacks sequences on chromosome 15. In scenario A, this results in a DSB in which the telomeric fragment of chromosome 15 is translocated to chromosome 12 while repair of the lesion on chromosome 15 occurs through a break-induced replication pathway such that sequences from chromosome 12 are copied to the der(15)a (steps 2a and 3a). In scenario B, c-myc is copied to der(12)b without disruption of the normal chromosome 15 (steps 2b and 3b). In both cases, however, the derivative chromosome is left with a persistent DSB. The broken chromatid is replicated during S-phase (step 4) and the free chromatid ends are ligated together to form a bridge (step 5). During mitosis when the chromatids are separated, a tug-of-war ensues resulting in the breaking of the derivative chromosome (step 6). Depending on where the break occurs, one cell may contain a chromosome with amplification of the genes as an inverted repeat (step 7). Because this chromosome still does not have a telomere sequence, the cycle continues until a telomere is captured (step 8).
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fig6: Proposed model for rearrangement events in the Kuβˆ’/βˆ’p53βˆ’/βˆ’ and Kuβˆ’/βˆ’p53βˆ’/βˆ’ Rad54βˆ’/βˆ’ pro-B cell lymphomas. RAG proteins initiate DNA DSB at the IgH D and J gene segments (step 1). Due to the absence of the Ku holoenzyme, the DSB cannot be efficiently and correctly repaired. The IgH DSB attacks sequences on chromosome 15. In scenario A, this results in a DSB in which the telomeric fragment of chromosome 15 is translocated to chromosome 12 while repair of the lesion on chromosome 15 occurs through a break-induced replication pathway such that sequences from chromosome 12 are copied to the der(15)a (steps 2a and 3a). In scenario B, c-myc is copied to der(12)b without disruption of the normal chromosome 15 (steps 2b and 3b). In both cases, however, the derivative chromosome is left with a persistent DSB. The broken chromatid is replicated during S-phase (step 4) and the free chromatid ends are ligated together to form a bridge (step 5). During mitosis when the chromatids are separated, a tug-of-war ensues resulting in the breaking of the derivative chromosome (step 6). Depending on where the break occurs, one cell may contain a chromosome with amplification of the genes as an inverted repeat (step 7). Because this chromosome still does not have a telomere sequence, the cycle continues until a telomere is captured (step 8).

Mentions: V(D)J recombination does, however, play a direct role in the development of Ku80βˆ’/βˆ’p53βˆ’/βˆ’ pro-B cell lymphomas as demonstrated by the proximity of the translocations on chromosome 12 to the JH loci and the absence of these tumors in Ku80βˆ’/βˆ’p53βˆ’/βˆ’RAG2βˆ’/βˆ’ mice. We thus demonstrate that RAG-induced DSBs at the IgH gene on chromosome 12 is the event initiating pro-B cell lymphomagenesis in Ku80βˆ’/βˆ’p53βˆ’/βˆ’ mice (Fig. 6 , step 1). In the majority of tumors (scenario A), a DSB is also generated telomeric to c-myc (Fig. 6, step 2a), as evidenced by the translocation of the distal portion of chromosome 15 to the der(12)a (Fig. 6, step 3a). Thus, at least two DSBs are generated in these tumors, an event which is predicted to increase the frequency of translocation by several orders of magnitude relative to a single DSB (1). Our analysis of the regions involved in the breakpoint on chromosome 15 reveals that they cluster ∼170–370 kb from the 3β€² end of c-myc in a region that appears devoid of recombination signal sequences. It is possible that the clustering reflects the presence of fragile sites in this region; alternatively, DNA cleavage on the partner chromosome may be targeted by RAG-mediated transposition (37, 38), an event that is stimulated by distorted DNA structures (39). Consistent with the latter possibility, the fusion points contained a combination of small inverted repeats and palindromic sequences (Fig. 4 A).


Evidence for replicative repair of DNA double-strand breaks leading to oncogenic translocation and gene amplification.

Difilippantonio MJ, Petersen S, Chen HT, Johnson R, Jasin M, Kanaar R, Ried T, Nussenzweig A - J. Exp. Med. (2002)

Proposed model for rearrangement events in the Kuβˆ’/βˆ’p53βˆ’/βˆ’ and Kuβˆ’/βˆ’p53βˆ’/βˆ’ Rad54βˆ’/βˆ’ pro-B cell lymphomas. RAG proteins initiate DNA DSB at the IgH D and J gene segments (step 1). Due to the absence of the Ku holoenzyme, the DSB cannot be efficiently and correctly repaired. The IgH DSB attacks sequences on chromosome 15. In scenario A, this results in a DSB in which the telomeric fragment of chromosome 15 is translocated to chromosome 12 while repair of the lesion on chromosome 15 occurs through a break-induced replication pathway such that sequences from chromosome 12 are copied to the der(15)a (steps 2a and 3a). In scenario B, c-myc is copied to der(12)b without disruption of the normal chromosome 15 (steps 2b and 3b). In both cases, however, the derivative chromosome is left with a persistent DSB. The broken chromatid is replicated during S-phase (step 4) and the free chromatid ends are ligated together to form a bridge (step 5). During mitosis when the chromatids are separated, a tug-of-war ensues resulting in the breaking of the derivative chromosome (step 6). Depending on where the break occurs, one cell may contain a chromosome with amplification of the genes as an inverted repeat (step 7). Because this chromosome still does not have a telomere sequence, the cycle continues until a telomere is captured (step 8).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196056&req=5

fig6: Proposed model for rearrangement events in the Kuβˆ’/βˆ’p53βˆ’/βˆ’ and Kuβˆ’/βˆ’p53βˆ’/βˆ’ Rad54βˆ’/βˆ’ pro-B cell lymphomas. RAG proteins initiate DNA DSB at the IgH D and J gene segments (step 1). Due to the absence of the Ku holoenzyme, the DSB cannot be efficiently and correctly repaired. The IgH DSB attacks sequences on chromosome 15. In scenario A, this results in a DSB in which the telomeric fragment of chromosome 15 is translocated to chromosome 12 while repair of the lesion on chromosome 15 occurs through a break-induced replication pathway such that sequences from chromosome 12 are copied to the der(15)a (steps 2a and 3a). In scenario B, c-myc is copied to der(12)b without disruption of the normal chromosome 15 (steps 2b and 3b). In both cases, however, the derivative chromosome is left with a persistent DSB. The broken chromatid is replicated during S-phase (step 4) and the free chromatid ends are ligated together to form a bridge (step 5). During mitosis when the chromatids are separated, a tug-of-war ensues resulting in the breaking of the derivative chromosome (step 6). Depending on where the break occurs, one cell may contain a chromosome with amplification of the genes as an inverted repeat (step 7). Because this chromosome still does not have a telomere sequence, the cycle continues until a telomere is captured (step 8).
Mentions: V(D)J recombination does, however, play a direct role in the development of Ku80βˆ’/βˆ’p53βˆ’/βˆ’ pro-B cell lymphomas as demonstrated by the proximity of the translocations on chromosome 12 to the JH loci and the absence of these tumors in Ku80βˆ’/βˆ’p53βˆ’/βˆ’RAG2βˆ’/βˆ’ mice. We thus demonstrate that RAG-induced DSBs at the IgH gene on chromosome 12 is the event initiating pro-B cell lymphomagenesis in Ku80βˆ’/βˆ’p53βˆ’/βˆ’ mice (Fig. 6 , step 1). In the majority of tumors (scenario A), a DSB is also generated telomeric to c-myc (Fig. 6, step 2a), as evidenced by the translocation of the distal portion of chromosome 15 to the der(12)a (Fig. 6, step 3a). Thus, at least two DSBs are generated in these tumors, an event which is predicted to increase the frequency of translocation by several orders of magnitude relative to a single DSB (1). Our analysis of the regions involved in the breakpoint on chromosome 15 reveals that they cluster ∼170–370 kb from the 3β€² end of c-myc in a region that appears devoid of recombination signal sequences. It is possible that the clustering reflects the presence of fragile sites in this region; alternatively, DNA cleavage on the partner chromosome may be targeted by RAG-mediated transposition (37, 38), an event that is stimulated by distorted DNA structures (39). Consistent with the latter possibility, the fusion points contained a combination of small inverted repeats and palindromic sequences (Fig. 4 A).

Bottom Line: Subsequent DNA repair events juxtaposing IgH and c-myc are mediated by a break-induced replication pathway.Cycles of breakage-fusion-bridge result in amplification of IgH/c-myc while chromosome stabilization occurs through telomere capture.Thus, mice deficient in NHEJ provide excellent models to study the etiology of unbalanced translocations and amplification events during tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. difilipm@mail.nih.gov

ABSTRACT
Nonreciprocal translocations and gene amplifications are commonly found in human tumors. Although little is known about the mechanisms leading to such aberrations, tissue culture models predict that they can arise from DNA breakage, followed by cycles of chromatid fusion, asymmetric mitotic breakage, and replication. Mice deficient in both a nonhomologous end joining (NHEJ) DNA repair protein and the p53 tumor suppressor develop lymphomas at an early age harboring amplification of an IgH/c-myc fusion. Here we report that these chromosomal rearrangements are initiated by a recombination activating gene (RAG)-induced DNA cleavage. Subsequent DNA repair events juxtaposing IgH and c-myc are mediated by a break-induced replication pathway. Cycles of breakage-fusion-bridge result in amplification of IgH/c-myc while chromosome stabilization occurs through telomere capture. Thus, mice deficient in NHEJ provide excellent models to study the etiology of unbalanced translocations and amplification events during tumorigenesis.

Show MeSH
Related in: MedlinePlus