Somatic structural rearrangements in genetically engineered mouse mammary tumors
Ignacio VarelaChristiaan KlijnP. J. StephensLaura MudieLucy StebbingsDanushka GalappaththigeHanneke van der GuldenEva SchutSjoerd KlarenbeekPeter J. CampbellLodewyk F.A. WesselsMichael R. StrattonJos JonkersP. Andrew FutrealDavid J. Adams
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Here we present the first paired-end sequencing of tumors from genetically engineered mouse models of cancer to determine how faithfully these models recapitulate the landscape of somatic rearrangements found in human tumors. These were models of Trp53-mutated breast cancer, Brca1- and Brca2-associated hereditary breast cancer, and E-cadherin (Cdh1) mutated lobular breast cancer. We show that although Brca1- and Brca2-deficient mouse mammary tumors have a defect in the homologous recombination pathway, there is no apparent difference in the type or frequency of somatic rearrangements found in these cancers when compared to other mouse mammary cancers, and tumors from all genetic backgrounds showed evidence of microhomology-mediated repair and non-homologous end-joining processes. Importantly, mouse mammary tumors were found to carry fewer structural rearrangements than human mammary cancers and expressed in-frame fusion genes. Like the fusion genes found in human mammary tumors, these were not recurrent. One mouse tumor was found to contain an internal deletion of exons of the Lrp1b gene, which led to a smaller in-frame transcript. We found internal in-frame deletions in the human ortholog of this gene in a significant number (4.2%) of human cancer cell lines. Paired-end sequencing of mouse mammary tumors revealed that they display significant heterogeneity in their profiles of somatic rearrangement but, importantly, fewer rearrangements than cognate human mammary tumors, probably because these cancers have been induced by strong driver mutations engineered into the mouse genome. Both human and mouse mammary cancers carry expressed fusion genes and conserved homozygous deletions.Keywords:
CDH1
Mammary tumor
Non-allelic homologous recombination
Non-homologous end joining
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Chromosomal crossover
Synapsis
Ectopic recombination
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Chromosomal crossover
Synapsis
Ectopic recombination
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Sexual reproduction prevails among eukaryotic organisms. The problem of advantage of sexual reproduction over asexual reproduction remains a subject of not stopping discussions. According to one of the hypotheses, sexual reproduction and homologous recombination which accompanies gamete formation during meiosis has arisen to increase genetic variability and, as consequence, a fitness of organisms. Many researches show that homologous recombination play an important role in reparation of DNA in various groups of organisms irrespective of the way of their reproduction. Involvement of recombination in meiosis, however, is impossible to explain only by DNA repair functions. The hypothesis, that a recombination in the course of sexual process is a source of variability, also is not capable to explain existence of this process well. There is convincing evidence that the homologous recombination in meiosis is necessary for formation of bivalents. A physical connection between homologous chromosomes that is formed by recombination is required for correct chromosome segregation during meiotic division and formation of gametes of full value.
Sexual reproduction
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Asexual reproduction
Chromosomal crossover
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Mitotic crossover
Gene conversion
FLP-FRT recombination
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In mammalian cells, the repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. Indirect evidence, including that from gene targeting and random integration experiments, had suggested that non-homologous mechanisms were significantly more frequent than homologous ones. However, more recent experiments indicate that homologous recombination is also a prominent DSB repair pathway. These experiments show that mammalian cells use homologous sequences located at multiple positions throughout the genome to repair a DSB. However, template preference appears to be biased, with the sister chromatid being preferred by 2–3 orders of magnitude over a homologous or heterologous chromosome. The outcome of homologous recombination in mammalian cells is predominantly gene conversion that is not associated with crossing-over. The preference for the sister chromatid and the bias against crossing-over seen in mitotic mammalian cells may have developed in order to reduce the potential for genome alterations that could occur when other homologous repair templates are utilized. In attempts to understand further the mechanism of homologous recombination, the proteins that promote this process are beginning to be identified. To date, four mammalian proteins have been demonstrated conclusively to be involved in DSB repair by homologous recombination: Rad54, XRCC2, XRCC3 and BRCAI. This paper summarizes results from a number of recent studies.
Non-homologous end joining
Homology directed repair
Branch migration
Non-allelic homologous recombination
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Abstract The number of clones that must be recovered will depend on a number of factors such as the type of selection (e.g. selection for homologous recombinants and against random integrants), number of colonies obtained after selection, method of screening for homologous recombinants (Southern vs PCR), knowledge about the targeting frequency for the locus of interest, etc. For obvious reasons, however, the more clones isolated the higher the probability of recovering a homologous recombinant. In situations in which we have targeted the same locus in multiple instances (e.g. the immunoglobulin heavy chain locus) we have generally observed a similar frequency of homologous recombination events and therefore know approximately how many colonies must be isolated to obtain a homologous recombinant. As a very general number, at least 250-300 colonies should be isolated for examination of a homologous recombination event.
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Non-allelic homologous recombination
Non-homologous end joining
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For targeted gene disruption in wild-type Neurospora crassa, 1000-bp of homologous sequences on either side of the cassette used for disruption is sufficient to give more than 10 % homologous recombination. We report here that varying the length of homology on each side seems to have different effects on the homologous recombination frequency.
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Crassa
Neurospora
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In mammalian cells, the repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. Indirect evidence, including that from gene targeting and random integration experiments, had suggested that non-homologous mechanisms were significantly more frequent than homologous ones. However, more recent experiments indicate that homologous recombination is also a prominent DSB repair pathway. These experiments show that mammalian cells use homologous sequences located at multiple positions throughout the genome to repair a DSB. However, template preference appears to be biased, with the sister chromatid being preferred by 2–3 orders of magnitude over a homologous or heterologous chromosome. The outcome of homologous recombination in mammalian cells is predominantly gene conversion that is not associated with crossing-over. The preference for the sister chromatid and the bias against crossing-over seen in mitotic mammalian cells may have developed in order to reduce the potential for genome alterations that could occur when other homologous repair templates are utilized. In attempts to understand further the mechanism of homologous recombination, the proteins that promote this process are beginning to be identified. To date, four mammalian proteins have been demonstrated conclusively to be involved in DSB repair by homologous recombination: Rad54, XRCC2, XRCC3 and BRCAI. This paper summarizes results from a number of recent studies.
Non-homologous end joining
Homology directed repair
Branch migration
Non-allelic homologous recombination
RAD52
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Strain YY1 and YY2 were two genetic recombinant strains derived from S.lincolnensis B48 by homologous exchange and homologous integration, respectively. Compared with B48, the potency of lincomycin of YY2 was much lower, while that of YY1 turned to be zero. The genetic recombination was further identified by PCR and the result showed that two copies of lmbI genes in YY1 were inactivated by homologous exchange, while probably only one copy in YY2 was inactivated by homologous integration and the other remained intact.
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