ZIA SC 010379 (ZIA) | |||
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Title | Mechanisms of Chromosomal Translocation | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Aplan, Peter | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $190,546 | Project Dates | 10/01/2001 - 00/00/0000 |
Fiscal Year | 2014 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Cancer (100.0%) Childhood Cancers (40.0%) |
Childhood Leukemia (40.0%) Leukemia (40.0%) Non Hodgkins Lymphoma (40.0%) Ovarian Cancer (20.0%) |
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Research Type | |||
Cancer Initiation: Alterations in Chromosomes Exogenous Factors in the origin and cause of cancer |
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Abstract | |||
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). The vast majority of the clones had small, interstitial deletions, and no clones showed GCR. However, 8 clones showed small DNA segments (50-1500 bp) that were derived from distant regions of the genome inserted at the DNA DSB site. Additional experiments recovered over 100 clones with insertions. Most of these insertions were either genic (transcribed) regions or repeat elements (LINE/SINE/LTR). These findings led to the hypothesis that these DNA DSB may have been repaired by ""patches"" generated from reverse transcribed RNA. To determine whether this form of DNA repair was restricted to experimentally induced DNA DSB, we analyzed whole genome sequence data from two myeloma cell lines, and identified 23 instances of insertions derived from distant genomic regions (which we have termed template sequence insertions, or TSIs). Most of these TSIs were seen in normal individuals as well as the myeloma cell lines, indicating that most TSIs were polymorphic in the human germline. Twelve examples showed the hallmarks of LINE-1 retrotransposon-mediated insertions, with a 5'-TTTT/A-3' integration site, a target site duplication (TSD), a polyA tract at the insertion site, and a polyadenylation signal. However, the inserted sequence was not a LINE-1 nor a SINE, but instead was a transcribed, genic region that mapped to a distant genomic region. The presence of these L1 ORF1 hallmarks strongly suggests that these TSIs were caused by the LINE-1 integrase and reverse-transcriptase acting on nuclear pre-mRNA. A second class of TSIs showed no preference for a 5'-TTTT/A-3' integration site, no TSD, no poly A tract, but instead showed several bp of microhomology at the insertion junction. This second class is remarkably similar to insertions at I-SceI induced DNA DSB, and we predict that these insertions were also caused by reverse transcription of pre-mRNA creating a ""patch"" for a spontaneous DNA DSB that occurred in a germ cell. We suspected that these TSIs could be templated by RNA intermediates, as the genomic regions were not lost from their wild-type chromosomal location. We repeated the I-SceI experiment several times, modified by co-transfection of mouse RNA with the I-SceI expression vector. Approximately 10% of the TSIs recovered from these experiments contained mouse sequences, indicating that they were derived from mouse RNA. In addition, a subset of TSIs were due to telomere repeat sequences, lending further support for the hypothesis that RNA could serve as a template for the repair of DNA DSB. 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 extraord" |