by David M. Weinstock, Beth Elliott, and Maria Jasin A model of oncogenic rearrangements: differences between chromosomal translocation mechanisms and simple double-strand break repair by David M. Weinstock, Beth Elliott, and Maria Jasin Blood Volume 107(2):777-780 January 15, 2006 ©2006 by American Society of Hematology

Chromosomal translocations between nonhomologous sequences. Chromosomal translocations between nonhomologous sequences. (A) Translocation design. A neo gene is split within its intron such that the 5′ portion with the splice donor (neoSD) is targeted to chromosome 17 in mouse ES cells, and the 3′ portion with the splice acceptor (SAneo) is targeted to chromosome 14. DSB induction at the I-SceI sites followed by interchromosomal NHEJ results in a neo+ gene on der(17). Because the breakpoint junction occurs within an intron, a variety of junctions can be recovered. The reciprocal chromosome der(14) also can form but is not under selection. Gray box, selectable marker. (B) Translocation reporters p5rE and p5rErev. The 2 rE alleles differ by whether the I-SceI sites on chromosomes 17 and 14 are in the same (rE) or opposite orientation (rErev). Der(17) arising from NHEJ with minimal sequence alteration reconstructs a neo+ gene with an intron of approximately 1.7 kb; der(14) can arise from NHEJ or SSA at the 210-bp repeat (green box), as shown. In principle, homology-directed repair at the 210-bp repeat could cause translocation formation via a reciprocal exchange. However, consistent with previous results,9-12 homology-directed repair events, which would leave one copy of the repeat on der(17), have not been recovered; hence, these translocation chromosomes are not diagrammed. Key distances are indicated in kb. (C) Translocation reporter pCr15. Der(17) arising from NHEJ with minimal sequence alteration reconstructs a neo+ gene with an intron of approximately 2.3 kb; like der(17), der(14) also can only arise from NHEJ. The I-SceI site is positioned further from the splice donor in the pCr15 reporter than in the p5rE and p5rErev reporters. For the pCr15 reporter, the I-SceI sites are in opposite orientation. The sequence of the I-SceI site upon cleavage is shown (blue box). (D) Fluorescence in situ hybridization using whole mouse chromosome 14-FITC (green) and chromosome 17-Cy3 (red) probes and demonstrating normal chromosomes 14 and 17 in the parental cell line and 2 derivative chromosomes (yellow arrows) in a neo+ clone. (E) Translocation chromosome analysis. PCR primers and sizes of PCR products are indicated on the derivative chromosomes in panel B. A sample of PCR products obtained from p5rE neo+ clones shows der(17) NHEJ junctions of variable length and der(14) NHEJ and SSA products (1.7 and 0.9 kb, respectively). No band is obtained from the parental cell line since the primers are located on different chromosomes. David M. Weinstock et al. Blood 2006;107:777-780 ©2006 by American Society of Hematology

Translocation junction analysis. Translocation junction analysis. (A) Distribution of deletion lengths in 172 translocation junctions formed by NHEJ for der(17) and der(14) junctions. Junctions are derived from neo+ clones from the 3 reporter cell lines. The der(14) junctions that are not shown here were repaired by SSA. NA indicates not applicable. (B) Scatter plot of translocation deletion lengths in which both derivative chromosome junctions in a neo+ clone arose by NHEJ. No correlation was noted between deletion lengths on der(17) and der(14) (r = -0.085). (C) Distribution of microhomologies in 138 translocation junctions. Only der(17) and der(14) junctions that were repaired by NHEJ without an insertion (ie, simple deletions) are included. David M. Weinstock et al. Blood 2006;107:777-780 ©2006 by American Society of Hematology

Comparison of translocation and intrachromosomal breakpoint junctions. Comparison of translocation and intrachromosomal breakpoint junctions. (A) Strategy to compare translocations and intrachromosomal repair at the same sequence. For translocations, I-SceI was expressed in the p5rE cell line, and for intrachromosomal repair, I-SceI was expressed in 2 p5rE translocation clones that re-established the I-SceI site on der(17) but not on der(14). After transfection, neo+ cells were selected in G418, and genomic DNA was digested with I-SceI prior to PCR amplification with the indicated primers. PCR products were cloned and sequenced. (B) Comparison of deletion lengths at translocation and intrachromosomal breakpoint junctions. The 53 intrachromosomal and 36 of the translocation junctions from p5rE der(17) were isolated by the PCR strategy shown in panel A; 38 translocation junctions from individually isolated p5rE neo+ clones that had not re-established the I-SceI site also are shown for der(17). (C) Comparison of microhomology distributions for translocation and intrachromosomal breakpoint junctions. The translocation junctions are compiled from the PCR strategy shown in panel A and neo+ clonal analysis for p5rE der(17). Only junctions containing simple deletions are included. David M. Weinstock et al. Blood 2006;107:777-780 ©2006 by American Society of Hematology