1 Nature 2004 Vol: 428(6978):88-93. DOI: 10.1038/nature02355

A non-B-DNA structure at the Bcl-2 major breakpoint region is cleaved by the RAG complex

The causes of spontaneous chromosomal translocations in somatic cells of biological organisms are largely unknown, although double-strand DNA breaks are required in all proposed mechanisms1, 2, 3, 4, 5. The most common chromosomal abnormality in human cancer is the reciprocal translocation between chromosomes 14 and 18 (t(14;18)), which occurs in follicular lymphomas. The break at the immunoglobulin heavy-chain locus on chromosome 14 is an interruption of the normal V(D)J recombination process. But the breakage on chromosome 18, at the Bcl-2 gene, occurs within a confined 150-base-pair region (the major breakpoint region or Mbr) for reasons that have remained enigmatic. We have reproduced key features of the translocation process on an episome that propagates in human cells. The RAG complex|[mdash]|which is the normal enzyme for DNA cleavage at V, D or J segments|[mdash]|nicks the Bcl-2 Mbr in vitro and in vivo in a manner that reflects the pattern of the chromosomal translocations; however, the Mbr is not a V(D)J recombination signal. Rather the Bcl-2 Mbr assumes a non-B-form DNA structure within the chromosomes of human cells at 20|[ndash]|30% of alleles. Purified DNA assuming this structure contains stable regions of single-strandedness, which correspond well to the translocation regions in patients. Hence, a stable non-B-DNA structure in the human genome appears to be the basis for the fragility of the Bcl-2 Mbr, and the RAG complex is able to cleave this structure.

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Figures
Figure 1: In vivo breakpoints on an extrachromosomal substrate cluster within the Bcl-2 Mbr and are dependent on the RAG complex.The episome (pSCR45) was transfected into mammalian cells (293T cells), recovered 40 h later and analysed in bacteria as described in the Methods. a, Comparison of breakpoint frequencies in recombinant plasmid molecules. DA, the number of substrate molecules that replicated in the 293T cells. Total DAC, bacterial transformants that are ampicillin–chloramphenical (double) resistant (also referred to as recombinants). The total DAC for each row is subdivided according to the left and right boundaries of recombination. For the left boundary, DACMbr is the subset of double-resistant recombinants that have a breakpoint within the Bcl-2 Mbr. DACNot Mbr is the subset of recombinants with breakpoints anywhere within the 326-bp region upstream or downstream of the Mbr. For the right boundary, DAC23 are recombinants that use the 23-signal sequence for recombination. DACNot 23 are recombinants that do not use the 23-signal for recombination. (DACMbr/DA) 100 is the recombination frequency for all events that use the Mbr. (DAC23/DA) 100 is the recombination frequency for all events that use the 23-signal. The frequency of recombinants obtained is indicated as a percentage (% Events). The row labelled 293 indicates recombinants obtained from 293T cells in the absence of RAGs; 293 + RAGs indicates that cells were also transfected with full-length RAG expression vectors; and 293 + Mut RAGs indicates that the cells were transfected with mutant RAG1 and full-length RAG-2 vectors. The asterisk indicates that this single event may represent a random break that just happens to be within the 13-bp region adjacent to the heptamer of the 23-signal8. b, Regions of recombination relative to the sequence of pSCR45. The Mbr region is shown in dark grey. The light grey regions are outside of the Mbr but are the flanking regions that naturally are adjacent to the Mbr in the human chromosome. The 12-signal is indicated by the open triangle, and the 23-signal by the filled triangle. The transcriptional promoter (short arrow upstream of the Mbr), the transcription terminator (Stop) and the chloramphenicol gene (Cat) are indicated. Figure 2: Bisulphite reactivity at the Bcl-2 Mbr on chromosomal DNA.A 528-bp fragment containing the Mbr amplified from chromosomal DNA after bisulphite treatment is shown. The 150-bp Bcl-2 Mbr region is expanded to show the complete sequence. The three breakpoint peaks are indicated by three short horizontal lines between the top and bottom strands (see Supplementary ). Bisulphite sensitivity is shown for the 250 bp downstream of the Mbr and 125 bp upstream of the Mbr. Vertical incremental bars (vertical dashes) above the line indicate the sensitivity for the top strand, and bars below the line indicate the sensitivity for the bottom strand; each vertical bar represents a cytosine conversion on one molecule. The total numbers of molecules sequenced from the top and bottom strands are indicated at the right margin. Figure 3: The bimodal nature (B-form DNA versus non-B-form) of the structural configuration of chromosomal DNA at the Mbr.The upper panel shows bisulphite sensitivity of molecules from the top strand, whereas the lower panel shows the corresponding bottom strand data. In both panels, each row of circles represents one DNA molecule. Each filled circle represents an instance of a bisulphite-converted cytosine (single-stranded), whereas open circles represent cytosines resistant to bisulphite (that is, double-stranded DNA). Note that only the C residues are depicted, but a scale to relate the length along the DNA is shown at the bottom. The designations I, II or III correspond to the three translocation frequency peaks within the Mbr. The population of molecules includes a mixture of those that are B-form and those that have substantial focal and recurring regions of single-strandedness (non-B-form). Figure 4: The bisulphite sensitivity of the Bcl-2 Mbr on plasmid DNA.The pXW5 plasmid DNA was purified from Escherichia coli by a non-denaturing method (see Supplementary Methods). Following bisulphite modification, a 930-bp DNA fragment containing the Bcl-2 Mbr was PCR-amplified and sequenced. a, b, Supercoiled plasmid DNA (a) and linear plasmid DNA (b) (linearized by BglII digestion) is shown. c, d, Distribution of bisulphite reactivity on molecules with seven or more C to T conversions in a string on the top (c) or bottom (d) strands of the Bcl-2 Mbr on supercoiled pXW5. (See Supplementary  legend for more details.)
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References
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    • . . . We transfected this cell line with a plasmid (pXW5) bearing the Bcl-2 Mbr (paired with a single 23-signal) with or without RAG1 and RAG2 expression vectors or with mutant RAG1 and RAG2 expression vectors8, 9 . . .
    • . . . The plasmid constructs were made by modifying the SV40-based plasmid, pGG51 (ref. 8) . . .
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    • . . . We transfected this cell line with a plasmid (pXW5) bearing the Bcl-2 Mbr (paired with a single 23-signal) with or without RAG1 and RAG2 expression vectors or with mutant RAG1 and RAG2 expression vectors8, 9 . . .
    • . . . The recombination frequency of pXW5 (0.004%) in the presence of RAGs is about 25-fold lower than that of a substrate with a pair of optimal 12/23-signals (recombination frequency of 0.1% in fibroblasts transfected with RAG vectors9) . . .
    • . . . Transfection of the 293T cells with pXW5 or pSCR45 along with full-length RAG1/2 (Fig. 1), mutant RAG1/full-length RAG2 (Fig. 1 and Table 1) or core RAG1/2 (Table 1) was done using the calcium-phosphate method as described earlier9 . . .
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    • . . . This plasmid, pSCR45, was then transfected into 293T cells with and without human full-length RAG expression vectors, or with a mutant RAG1 vector10. . . .
    • . . . Core murine glutathione S-transferase (GST)|[ndash]|RAG1 (amino acids 330|[ndash]|1040) and GST|[ndash]|RAG2 (amino acids 1|[ndash]|383), or MBP murine core RAG1 and RAG2, or core RAG1 and full-length MBP RAG2 proteins were overexpressed in the human 293T cells and purified as previously described10, 18 . . .
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    • . . . To test for single-strandedness of the Bcl-2 Mbr, we used chemical-probing methods on genomic DNA11, 12 . . .
    • . . . Bisulphite converts unpaired cytosines to uracil, and these become thymine after PCR amplification, thus allowing the detection of single-stranded regions11. . . .
    • . . . The bisulphite modification assay was used as described previously11 (see Supplementary Methods). . . .
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    • . . . To test for single-strandedness of the Bcl-2 Mbr, we used chemical-probing methods on genomic DNA11, 12 . . .
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    • . . . The simplest explanation for this is that one or both of the coding ends are released during a 12/23 paired V(D)J recombination event13 and simultaneously a break at the Bcl-2 Mbr is generated by the RAGs . . .
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    • . . . First, the RAG complex (and other transposase enzymes) cleaves single-stranded 3|[prime]| overhangs in Mg2|[plus]|-containing buffers14 . . .
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    • . . . This may reflect some common structural features between 3|[prime]| overhangs and the presumed single-strandedness thought to exist at the border of the heptamer, where RAGs normally cleave15 . . .
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    • . . . Second, the RAG complex opens DNA hairpins in Mn2|[plus]|-containing buffers16 . . .
  17. Ma, Y., Pannicke, U., Schwarz, K. & Lieber, M. R. Hairpin opening and overhang processing by an Artemis:DNA-PKcs complex in V(D)J recombination and in nonhomologous end joining. Cell 108, 781−794 , .
    • . . . This RAG-mediated hairpin opening is inefficient in Mg2|[plus]| buffers, and it may not be physiologically relevant to hairpin opening17, but it illustrates that RAG proteins have some low level of activity on such non-B configurations . . .
  18. Yu, K. & Lieber, M. R. The nicking step of V(D)J recombination is independent of synapsis: implications for the immune repertoire. Mol. Cell. Biol. 20, 7914−7921 , .
    • . . . Core murine glutathione S-transferase (GST)|[ndash]|RAG1 (amino acids 330|[ndash]|1040) and GST|[ndash]|RAG2 (amino acids 1|[ndash]|383), or MBP murine core RAG1 and RAG2, or core RAG1 and full-length MBP RAG2 proteins were overexpressed in the human 293T cells and purified as previously described10, 18 . . .
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