To develop a model system in which we could induce GCRs, we used the rare restriction enzyme I-SceI, whose 18 bp recognition sequence is not normally present in the human or mouse genome, to produce a single DNA DSB within a mammalian cell, based on the hypothesis that improper repair of these breaks could lead to GCRs. This enzyme has been used in a series of elegant studies to produce specific, non-random GCRs mediated by homologous recombination in mammalian cells. We generated a construct that expressed the Herpes simplex virus type I thymidine kinase (TK) gene under the control of the constitutive EF1a promoter, with the recognition sequence for the I-SceI restriction enzyme placed between the EF1a promoter and the TK gene. This pEF1aTK vector was introduced into the U937 cell line, and verified that expression of the TK gene conferred sensitivity to ganciclovir (GCV). We then carried out a series of experiments that utilized the negative selection provided by the expression of TK. Cells were transfected with an I-SceI expression vector and selected with GCV (to select for cells that had lost TK expression). All 156 of the clones had small deletions and showed evidence consistent with non-homologous end joining (NHEJ), such as microhomology or local sequence inversions. No clones showed evidence of having a GCR. It may not be surprising that we were unable to generate GCRs by inducing a single DNA DSB, as other investigators have concluded that two induced breaks are required to produce a chromosomal translocation, and that the frequency of chromosomal translocations induced by a single DNA DSB in mouse embryonic stem (ES) cells is extraordinarily rare (2-fold greater in the Pu/Py clones compared to the F5 clone described above, we recovered no GCR. However, 19 of the 25 Pu/Py clones evaluated showed spontaneous mutations (single nucleotide replacements or frameshifts) of the Tk gene, compared to only 2 of 20 F5 clones evaluated, suggesting that Pu/Py sequence predisposed the Tk sequence to spontaneous mutation. We are also attempting to move this system from a cell-culture based system to an in vivo system. To do so, we take advantage of the observation that the MLL gene is known to have at least 100 oncogenic partners, and is even weakly oncogenic when fused to a LacZ reporter gene. These findings suggest that MLL fused to many other genes may be oncogenic. We used gene targeting to insert an I-SceI site in the MLL (MLLKI) locus. These mice have been crossed to mice that express the ISceI (vav-I) protein in hematopoietic cells, to test the possibility that a GCR involving MLL will be generated in vivo. We reasoned that even if this is a very rare event, it may be amplified in vivo, as cells that undergo an MLL fusion may be oncogenic. Unfortunately, none of over 40 MLLKI/vav-I mice developed leukemia. Analysis of bone marrow cells from the MLLKI/vav-I mice indicated that the I-SceI enzyme was active, as there were cells with small (1-10 bp) deletions or insertions at the I-SceI site. We reasoned that no leukemias may have been generated because repair of the I-SceI induced breaks is very efficient. We have now begun to cross these transgenes onto an H2AX deficient background, to determine if a more error-prone DNA repair pathway will result in MLL translocations in vivo.

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National Cancer Institute (NCI)
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National Cancer Institute Division of Clinical Sciences
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Qiu, Zhijun; Zhang, Zhenhua; Roschke, Anna et al. (2017) Generation of Gross Chromosomal Rearrangements by a Single Engineered DNA Double Strand Break. Sci Rep 7:43156
Canela, Andres; Maman, Yaakov; Jung, Seolkyoung et al. (2017) Genome Organization Drives Chromosome Fragility. Cell 170:507-521.e18
Onozawa, Masahiro; Aplan, Peter D (2016) Templated Sequence Insertion Polymorphisms in the Human Genome. Front Chem 4:43
Onozawa, Masahiro; Goldberg, Liat; Aplan, Peter D (2015) Landscape of insertion polymorphisms in the human genome. Genome Biol Evol 7:960-8
Onozawa, Masahiro; Zhang, Zhenhua; Kim, Yoo Jung et al. (2014) Repair of DNA double-strand breaks by templated nucleotide sequence insertions derived from distant regions of the genome. Proc Natl Acad Sci U S A 111:7729-34
Onozawa, Masahiro; Aplan, Peter D (2012) Illegitimate V(D)J recombination involving nonantigen receptor loci in lymphoid malignancy. Genes Chromosomes Cancer 51:525-35
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