Gene Conversion Tracts from Double-Strand Break Repair in Mammalian Cells

Elliott, Beth; Richardson, Christine; Winderbaum, Jamie; Nickoloff, Jac A.; Jasin, Maria

doi:10.1128/mcb.18.1.93

Cited by 294 publications

(253 citation statements)

References 52 publications

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“…Genetic heterology can reduce the recombination frequency in bacteria (Shen and Huang 1986), yeast Hunter et al 1996), and mice (Elliott et al 1998;Spies and Fishel 2015) via mechanisms mediated by the mismatch repair machinery (Rayssiguier et al 1989). Genetic crossovers are one outcome of successful recombination, and, indeed, by comparing crossovers with DSB hot spots in mouse hybrids, we found that crossovers occur preferentially at sites with below average genetic divergence (Fig.…”

Section: Discussionmentioning

confidence: 98%

The evolutionary turnover of recombination hot spots contributes to speciation in mice

et al. 2016

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Meiotic recombination is required for the segregation of homologous chromosomes and is essential for fertility. In most mammals, the DNA double-strand breaks (DSBs) that initiate meiotic recombination are directed to a subset of genomic loci (hot spots) by sequence-specific binding of the PRDM9 protein. Rapid evolution of the DNA-binding specificity of PRDM9 and gradual erosion of PRDM9-binding sites by gene conversion will alter the recombination landscape over time. To better understand the evolutionary turnover of recombination hot spots and its consequences, we mapped DSB hot spots in four major subspecies of Mus musculus with different Prdm9 alleles and in their F1 hybrids. We found that hot spot erosion governs the preferential usage of some Prdm9 alleles over others in hybrid mice and increases sequence diversity specifically at hot spots that become active in the hybrids. As crossovers are disfavored at such hot spots, we propose that sequence divergence generated by hot spot turnover may create an impediment for recombination in hybrids, potentially leading to reduced fertility and, eventually, speciation.

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Section: Discussionmentioning

confidence: 98%

The evolutionary turnover of recombination hot spots contributes to speciation in mice

et al. 2016

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“…However rapid and efficient, gene correction is a localized event, and a single DSB, whether induced by a homing endonuclease (26) or by a ZFN (M.C.H., Y.-L.L., and F.D.U., unpublished data), can allow efficient correction of mutations only within an Ϸ200-bp window surrounding the break. The complex mutational spectrum underlying many human monogenic diseases would therefore require tailoring ZFNs to each cluster of mutations.…”

mentioning

confidence: 99%

Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases

Moehle

Rock

Lee

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

342

253

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Efficient incorporation of novel DNA sequences into a specific site in the genome of living human cells remains a challenge despite its potential utility to genetic medicine, biotechnology, and basic research. We find that a precisely placed double-strand break induced by engineered zinc finger nucleases (ZFNs) can stimulate integration of long DNA stretches into a predetermined genomic location, resulting in high-efficiency site-specific gene addition. Using an extrachromosomal DNA donor carrying a 12-bp tag, a 900-bp ORF, or a 1.5-kb promoter-transcription unit flanked by locus-specific homology arms, we find targeted integration frequencies of 15%, 6%, and 5%, respectively, within 72 h of treatment, and with no selection for the desired event. Importantly, we find that the integration event occurs in a homology-directed manner and leads to the accurate reconstruction of the donor-specified genotype at the endogenous chromosomal locus, and hence presumably results from synthesis-dependent strand annealing repair of the break using the donor DNA as a template. This site-specific gene addition occurs with no measurable increase in the rate of random integration. Remarkably, we also find that ZFNs can drive the addition of an 8-kb sequence carrying three distinct promoter-transcription units into an endogenous locus at a frequency of 6%, also in the absence of any selection. These data reveal the surprising versatility of the specialized polymerase machinery involved in double-strand break repair, illuminate a powerful approach to mammalian cell engineering, and open the possibility of ZFN-driven gene addition therapy for human genetic disease.

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“…We cannot rule this out, because the distance between the heterozygous variants in intron 23 and exon 30 flanking the maternal mutation in exon 29 (see Fig. 1) was approximate 12 kb (31), whereas the most common geneconversion tract lengths demonstrated in an experimental mammalian system were 58 bp or less (32). However, spontaneous gene-conversion events are rare, and slipped-strand mispairing, which is thought to be responsible for a significant proportion of FANCA polymorphism screening in flow-sorted colonies.…”

Section: Discussionmentioning

confidence: 99%

Somatic mosaicism in Fanconi anemia: Evidence of genotypic reversion in lymphohematopoietic stem cells

Gregory

Wagner

Verlander

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

199

137

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Somatic mosaicism has been observed previously in the lymphocyte population of patients with Fanconi anemia (FA). To identify the cellular origin of the genotypic reversion, we examined each lymphohematopoietic and stromal cell lineage in an FA patient with a 2815-2816ins19 mutation in FANCA and known lymphocyte somatic mosaicism. DNA extracted from individually plucked peripheral blood T cell colonies and marrow colony-forming unit granulocyte-macrophage and burst-forming unit erythroid cells revealed absence of the maternal FANCA exon 29 mutation in 74.0%, 80.3%, and 86.2% of colonies, respectively. These data, together with the absence of the FANCA exon 29 mutation in Epstein-Barr virus-transformed B cells and its presence in fibroblasts, indicate that genotypic reversion, most likely because of back mutation, originated in a lymphohematopoietic stem cell and not solely in a lymphocyte population. Contrary to a predicted increase in marrow cellularity resulting from reversion in a hematopoietic stem cell, pancytopenia was progressive. Additional evaluations revealed a partial deletion of 11q in 3 of 20 bone marrow metaphase cells. By using interphase fluorescence in situ hybridization with an MLL gene probe mapped to band 11q23 to identify colonyforming unit granulocyte-macrophage and burst-forming unit erythroid cells with the 11q deletion, the abnormal clone was exclusive to colonies with the FANCA exon 29 mutation. Thus, we demonstrate the spontaneous genotypic reversion in a lymphohematopoietic stem cell. The subsequent development of a clonal cytogenetic abnormality in nonrevertant cells suggests that ex vivo correction of hematopoietic stem cells by gene transfer may not be sufficient for providing life-long stable hematopoiesis in patients with FA. F anconi anemia (FA) is an autosomal recessive disorder characterized by congenital abnormalities, bone marrow failure, and a predisposition to malignancies, including myelodysplastic syndrome and acute myelogenous leukemia (1). Seven FA complementation groups (FA-A through FA-G) have been reported (2), with FA-A (Online Mendelian Inheritance in Man, OMIM no. 227650) constituting approximately two-thirds of the patients. The FANCA, FANCC, FANCE, FANCF, and FANCG genes have been cloned and mapped to chromosomes 16q24. 3, 9q22.3, 6p21.2-21.3, 11p15, and 9p13, respectively (3-8). However, the specific function of these genes remains unclear.FA cells are uniquely hypersensitive to DNA cross-linking agents such as diepoxybutane (DEB) and mitomycin C (MMC) (9). This cellular phenotype can be demonstrated in cultured T cells, B cells, fibroblasts, and fetal cells cultured from amniotic fluid or chorionic villi (10). Hypersensitivity of phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBLs) to DEB is used routinely as a diagnostic test for the syndrome (11). At a DEB concentration that has a minimal effect on normal PBLs (0.1 g͞ml), 80-100% of PBLs analyzed will have chromatid breaks and exchanges in the typical FA patient. However, in a small grou...

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Gene Conversion Tracts from Double-Strand Break Repair in Mammalian Cells

Cited by 294 publications

References 52 publications

The evolutionary turnover of recombination hot spots contributes to speciation in mice

The evolutionary turnover of recombination hot spots contributes to speciation in mice

Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases

Somatic mosaicism in Fanconi anemia: Evidence of genotypic reversion in lymphohematopoietic stem cells

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