Deficiency of the Fanconi anemia E2 ubiqitin conjugase UBE2T only partially abrogates Alu-mediated recombination in a new model of homology dependent recombination
| Autor: | René M. Linka, Todd Lewis, Paul R. Andreassen, Joanna Barthelemy, Feng Zhang, Elizabeth L. Virts, Felicia M. Kennedy, Rujuta Yashodhan Gadgil, Constanze Wiek, Michael Leffak, Helmut Hanenberg |
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| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
Medizin
Reversion Alu element Biology Genome Integrity Repair and Replication 03 medical and health sciences 0302 clinical medicine Fanconi anemia Alu Elements Gene Duplication Gene duplication Genetics Null cell medicine Humans DNA Breaks Double-Stranded Homologous Recombination Gene 030304 developmental biology 0303 health sciences Fanconi Anemia Complementation Group D2 Protein medicine.disease Hematopoietic Stem Cells Molecular biology Fanconi Anemia Complementation Group Proteins 3. Good health Fanconi Anemia Ubiquitin-Conjugating Enzymes Paternal Inheritance Maternal Inheritance Stem cell CRISPR-Cas Systems Homologous recombination 030217 neurology & neurosurgery Gene Deletion DNA Damage HeLa Cells |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| Popis: | The primary function of the UBE2T ubiquitin conjugase is in the monoubiquitination of the FANCI-FANCD2 heterodimer, a central step in the Fanconi anemia (FA) pathway. Genetic inactivation of UBE2T is responsible for the phenotypes of FANCT patients; however, a FANCT patient carrying a maternal duplication and a paternal deletion in the UBE2T loci displayed normal peripheral blood counts and UBE2T protein levels in B-lymphoblast cell lines. To test whether reversion by recombination between UBE2T AluYa5 elements could have occurred in the patient's hematopoietic stem cells despite the defects in homologous recombination (HR) in FA cells, we constructed HeLa cell lines containing the UBE2T AluYa5 elements and neighboring intervening sequences flanked by fluorescent reporter genes. Introduction of a DNA double strand break in the model UBE2T locus in vivo promoted single strand annealing (SSA) between proximal Alu elements and deletion of the intervening color marker gene, recapitulating the reversion of the UBE2T duplication in the FA patient. To test whether UBE2T null cells retain HR activity, the UBE2T genes were knocked out in HeLa cells and U2OS cells. CRISPR/Cas9-mediated genetic knockout of UBE2T only partially reduced HR, demonstrating that UBE2T-independent pathways can compensate for the recombination defect in UBE2T/FANCT null cells. CA extern |
| Databáze: | OpenAIRE |
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