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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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Sequence analysis and localization of fusion points and rearrangements in the IgH Ca and c-myc loci. (A) The fusion points of three Ku80−/−p53−/− pro-B cell lymphomas (PKT no. 2, 7, and 13) were cloned by digestion circularization PCR [der(15)a]. Sequence analysis of the three breakpoints from scenario A demonstrate microhomology (yellow) at the junction of chromosomes 12 (red) and 15 (green) in two cases (PKT2 and PKT7), but none in tumor PKT13. Homologous bases outside of this region are also indicated in uppercase letters. (B) Sequence analysis implied that the breakpoint was the V(D)J recombination signal sequence (RSS; blue) flanking IgH J1 in PKT2 and IgH J4 in PKT7 and PKT13 (top panel); however, exonuclease digestion of the cleaved ends resulted in loss of 45, 208, and 189 bp, respectively, from the original site of the RAG-mediated DSB to the fusion point (arrows). Approximate localization of the breakpoints on chromosome 15 (second panel) was determined by FISH hybridization with two BAC clones in this region (96F18 and 212H11) as illustrated in Fig. 5. Tumors 1–4, 7, and 13 are from Ku80−/−p53−/− mice, tumors *1–*3 are from Ku70−/−p53−/− mice and tumors N9984 and N10527 are from Ku80−/−p53−/−RAD54−/− mice. The resulting der(15)a and der(12)b chromosomes are illustrated in the bottom panels. In scenario A a portion of the IgH locus is copied to chromosome 15 downstream of c-myc and in the reverse orientation. In scenario B it appears that the copying of chromosome 15 to the der(12)b initiates centromeric of the c-myc locus and continues along toward the telomere.
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fig4: Sequence analysis and localization of fusion points and rearrangements in the IgH Ca and c-myc loci. (A) The fusion points of three Ku80−/−p53−/− pro-B cell lymphomas (PKT no. 2, 7, and 13) were cloned by digestion circularization PCR [der(15)a]. Sequence analysis of the three breakpoints from scenario A demonstrate microhomology (yellow) at the junction of chromosomes 12 (red) and 15 (green) in two cases (PKT2 and PKT7), but none in tumor PKT13. Homologous bases outside of this region are also indicated in uppercase letters. (B) Sequence analysis implied that the breakpoint was the V(D)J recombination signal sequence (RSS; blue) flanking IgH J1 in PKT2 and IgH J4 in PKT7 and PKT13 (top panel); however, exonuclease digestion of the cleaved ends resulted in loss of 45, 208, and 189 bp, respectively, from the original site of the RAG-mediated DSB to the fusion point (arrows). Approximate localization of the breakpoints on chromosome 15 (second panel) was determined by FISH hybridization with two BAC clones in this region (96F18 and 212H11) as illustrated in Fig. 5. Tumors 1–4, 7, and 13 are from Ku80−/−p53−/− mice, tumors *1–*3 are from Ku70−/−p53−/− mice and tumors N9984 and N10527 are from Ku80−/−p53−/−RAD54−/− mice. The resulting der(15)a and der(12)b chromosomes are illustrated in the bottom panels. In scenario A a portion of the IgH locus is copied to chromosome 15 downstream of c-myc and in the reverse orientation. In scenario B it appears that the copying of chromosome 15 to the der(12)b initiates centromeric of the c-myc locus and continues along toward the telomere.

Mentions: The IgH locus in the mouse is oriented such that transcription initiates at the telomeric V regions and proceeds downstream toward the more centromeric constant regions (see Fig. 4) . In Ku80−/−p53−/− lymphomas, chromosome 15 was translocated to chromosome 12 upstream of the JH segments while the more telomeric variable (VH) region was lost, suggesting that these events were initiated by Rag-mediated cleavage (4). To directly test the requirement for V(D)J recombination in the generation of pro-B cell lymphomas, we generated Ku80−/−p53−/−RAG2−/− mice. These mice did not succumb to pro-B cell lymphomas, but unexpectedly, developed thymic lymphomas at approximately the same time (3 mo) that pro-B cell lymphomas developed in Ku80−/−p53−/− mice (Table I). Chromosome aberrations in Ku80−/−p53−/−RAG2−/− thymomas were similar to those reported for p53−/− thymomas in that they exhibited polyploidy and aneuploidy, with very few translocations detectable by SKY analysis (not shown). We conclude that Rag-mediated cleavage is essential for the development of pro-B cell lymphomas in Ku80−/−p53−/− mice, but it is dispensable for the development of thymomas in Ku80−/−p53−/−Rag2−/− mice. Consistent with these results, no B-lineage tumors were observed in SCIDp53−/−Rag2−/− mice of which 43% developed thymic lymphomas (12).


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)

Sequence analysis and localization of fusion points and rearrangements in the IgH Ca and c-myc loci. (A) The fusion points of three Ku80−/−p53−/− pro-B cell lymphomas (PKT no. 2, 7, and 13) were cloned by digestion circularization PCR [der(15)a]. Sequence analysis of the three breakpoints from scenario A demonstrate microhomology (yellow) at the junction of chromosomes 12 (red) and 15 (green) in two cases (PKT2 and PKT7), but none in tumor PKT13. Homologous bases outside of this region are also indicated in uppercase letters. (B) Sequence analysis implied that the breakpoint was the V(D)J recombination signal sequence (RSS; blue) flanking IgH J1 in PKT2 and IgH J4 in PKT7 and PKT13 (top panel); however, exonuclease digestion of the cleaved ends resulted in loss of 45, 208, and 189 bp, respectively, from the original site of the RAG-mediated DSB to the fusion point (arrows). Approximate localization of the breakpoints on chromosome 15 (second panel) was determined by FISH hybridization with two BAC clones in this region (96F18 and 212H11) as illustrated in Fig. 5. Tumors 1–4, 7, and 13 are from Ku80−/−p53−/− mice, tumors *1–*3 are from Ku70−/−p53−/− mice and tumors N9984 and N10527 are from Ku80−/−p53−/−RAD54−/− mice. The resulting der(15)a and der(12)b chromosomes are illustrated in the bottom panels. In scenario A a portion of the IgH locus is copied to chromosome 15 downstream of c-myc and in the reverse orientation. In scenario B it appears that the copying of chromosome 15 to the der(12)b initiates centromeric of the c-myc locus and continues along toward the telomere.
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Related In: Results  -  Collection

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fig4: Sequence analysis and localization of fusion points and rearrangements in the IgH Ca and c-myc loci. (A) The fusion points of three Ku80−/−p53−/− pro-B cell lymphomas (PKT no. 2, 7, and 13) were cloned by digestion circularization PCR [der(15)a]. Sequence analysis of the three breakpoints from scenario A demonstrate microhomology (yellow) at the junction of chromosomes 12 (red) and 15 (green) in two cases (PKT2 and PKT7), but none in tumor PKT13. Homologous bases outside of this region are also indicated in uppercase letters. (B) Sequence analysis implied that the breakpoint was the V(D)J recombination signal sequence (RSS; blue) flanking IgH J1 in PKT2 and IgH J4 in PKT7 and PKT13 (top panel); however, exonuclease digestion of the cleaved ends resulted in loss of 45, 208, and 189 bp, respectively, from the original site of the RAG-mediated DSB to the fusion point (arrows). Approximate localization of the breakpoints on chromosome 15 (second panel) was determined by FISH hybridization with two BAC clones in this region (96F18 and 212H11) as illustrated in Fig. 5. Tumors 1–4, 7, and 13 are from Ku80−/−p53−/− mice, tumors *1–*3 are from Ku70−/−p53−/− mice and tumors N9984 and N10527 are from Ku80−/−p53−/−RAD54−/− mice. The resulting der(15)a and der(12)b chromosomes are illustrated in the bottom panels. In scenario A a portion of the IgH locus is copied to chromosome 15 downstream of c-myc and in the reverse orientation. In scenario B it appears that the copying of chromosome 15 to the der(12)b initiates centromeric of the c-myc locus and continues along toward the telomere.
Mentions: The IgH locus in the mouse is oriented such that transcription initiates at the telomeric V regions and proceeds downstream toward the more centromeric constant regions (see Fig. 4) . In Ku80−/−p53−/− lymphomas, chromosome 15 was translocated to chromosome 12 upstream of the JH segments while the more telomeric variable (VH) region was lost, suggesting that these events were initiated by Rag-mediated cleavage (4). To directly test the requirement for V(D)J recombination in the generation of pro-B cell lymphomas, we generated Ku80−/−p53−/−RAG2−/− mice. These mice did not succumb to pro-B cell lymphomas, but unexpectedly, developed thymic lymphomas at approximately the same time (3 mo) that pro-B cell lymphomas developed in Ku80−/−p53−/− mice (Table I). Chromosome aberrations in Ku80−/−p53−/−RAG2−/− thymomas were similar to those reported for p53−/− thymomas in that they exhibited polyploidy and aneuploidy, with very few translocations detectable by SKY analysis (not shown). We conclude that Rag-mediated cleavage is essential for the development of pro-B cell lymphomas in Ku80−/−p53−/− mice, but it is dispensable for the development of thymomas in Ku80−/−p53−/−Rag2−/− mice. Consistent with these results, no B-lineage tumors were observed in SCIDp53−/−Rag2−/− mice of which 43% developed thymic lymphomas (12).

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