Abstract
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.
Highlights
Alu elements are the most abundant short interspersed elements (SINEs) in the human genome, numbering over one million copies
Using the dual fluorescence system, we find that UBE2T has a limited role in homologous recombination (HR), and demonstrate the role of homology directed recombination (HDR) in Alu-mediated recombination in UBE2T using inhibitors or knockdown of HDR or nonhomologous end joining (NHEJ)-related proteins
The E2 ubiquitin conjugases UBE2T and UBE2W can act in the ubiquitination of FANCI/D2
Summary
Alu elements are the most abundant short interspersed elements (SINEs) in the human genome, numbering over one million copies These repetitive sequences are hotspots for genetic intrachromosomal or interchromosomal recombination [1]. Alumediated recombination (AMR) events contribute to multiple forms of cancer and other genetic disorders [3,4,5,6,7,8], and are estimated to be responsible for 0.3% of human genetic diseases [4,9] These repeated elements drive genomic evolution; it has been estimated that more than five hundred Alu-mediated deletion events have occurred since divergence of the human and chimpanzee genomes [9]. In the current model system, an in vivo double strand break leads to homology-dependent recombination between two UBE2T Alu elements, mimicking a contraction of the maternal duplication to restore the WT allele
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