PubMed · 10561839
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K M Vasquez, J H Wilson. 2000. Triplex-directed site-specific genome modification.. https://doi.org/10.1385/1-59259-215-5%3A183
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We have shown previously that a wide range of mutagenic carcinogens are capable of inducing loss of heterozygosity (LOH) at the endogenous Aprt locus in mouse splenic lymphocytes. To investigate whether LOH might be caused by a single common mechanism, we set out to determine the extent of LOH by microsatellite analysis along (the Aprt gene containing) mouse chromosome 8. Aprt+/- hybrid B6C3F1 mice were treated with mutagens that induce different classes of DNA lesions, i.e. bulky DNA adducts, DNA methylation, DNA inter-strand crosslinks or DNA strand breaks. Aprt mutant frequencies (MF) in this C57Bl/6-C3H hybrid background were significantly reduced for mitomycin C (MMC) and methylmethanesulfonate (MMS) in comparison with MF in C57Bl/6 background, suggesting either enhanced repair or reduced formation of MMC- or MMS-induced mutagenic lesions in a hybrid B6C3F1 background. In contrast, Aprt MF after dimethylbenz[a]anthracene (DMBA), methylnitrosurea (MNU) and etoposide treatment were similar in both genetic backgrounds. Microsatellite analysis of Aprt mutant clones indicated a dominant role for mitotic recombination (MR) in generating spontaneous, DMBA- and etoposide-induced LOH at APRT: However, over 80% of the MMC-induced Aprt LOH mutants had lost heterozygosity for all markers tested, suggesting that either the crossover points were located close to the centromere or that these mutants arose by chromosome loss and duplication of the remaining chromosome 8. A substantial fraction (40%) of MNU-induced Aprt mutants had lost the wild-type Aprt allele, but had retained heterozygosity at all polymorphic markers tested at chromosome 8 indicating an important role for deletions in LOH formation by MNU. Patterns of MR differed quite dramatically for the various chemical mutagens tested, suggesting different mechanisms to be involved in inducing recombination between homologous chromosomes. In addition, non-random adduct formation and repair between chromosomal regions, i.e. heterochromatin versus euchromatin, may contribute to a non-random distribution of recombinational crossover points.
Expansion of CTG triplet repeats in the 3' untranslated region of the DMPK gene causes the autosomal dominant disorder myotonic dystrophy. Instability of CTG repeats is thought to arise from their capacity to form hairpin DNA structures. How these structures interact with various aspects of DNA metabolism has been studied intensely for Escherichia coli and Saccharomyces cerevisiae but is relatively uncharacterized in mammalian cells. To examine the stability of (CTG)(17), (CTG)(98), and (CTG)(183) repeats during homologous recombination, we placed them in the second intron of one copy of a tandemly duplicated pair of APRT genes. Cells selected for homologous recombination between the two copies of the APRT gene displayed distinctive patterns of change. Among recombinants from cells with (CTG)(98) and (CTG)(183), 5% had lost large numbers of repeats and 10% had suffered rearrangements, a frequency more than 50-fold above normal levels. Analysis of individual rearrangements confirmed the involvement of the CTG repeats. Similar changes were not observed in proliferating (CTG)(98) and (CTG)(183) cells that were not recombinant at APRT. Instead, they displayed high frequencies of small changes in repeat number. The (CTG)(17) repeats were stable in all assays. These studies indicate that homologous recombination strongly destabilizes long tracts of CTG repeats.
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