PML isoform expression and DNA break location relative to PML nuclear bodies impacts the efficiency of homologous recombination
| Autor: | Graham Dellaire, Sabateeshan Mathavarajah, Carter Van Iderstine, Kathleen M. Attwood, Jayme Salsman, Dudley Chung |
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| Rok vydání: | 2019 |
| Předmět: |
Gene isoform
viruses Genotoxic Stress Promyelocytic Leukemia Protein Biochemistry Homology directed repair 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Cell Line Tumor medicine CRISPR Humans Protein Isoforms DNA Breaks Double-Stranded Homologous Recombination Molecular Biology In Situ Hybridization Fluorescence 030304 developmental biology Cell Nucleus 0303 health sciences virus diseases Recombinational DNA Repair Cell Biology medicine.disease Chromatin Cell biology Leukemia chemistry Microscopy Fluorescence 030220 oncology & carcinogenesis CRISPR-Cas Systems Homologous recombination DNA |
| Zdroj: | Biochemistry and cell biology = Biochimie et biologie cellulaire. 98(3) |
| ISSN: | 1208-6002 |
| Popis: | Promyelocytic leukemia nuclear bodies (PML NBs) are nuclear subdomains that respond to genotoxic stress by increasing in number via changes in chromatin structure. However, the role of the PML protein and PML NBs in specific mechanisms of DNA repair has not been fully characterized. Here, we have directly examined the role of PML in homologous recombination (HR) using I-SceI extrachromosomal and chromosome-based homology-directed repair (HDR) assays, and in HDR by CRISPR/Cas9-mediated gene editing. We determined that PML loss can inhibit HR in an extrachromosomal HDR assay but had less of an effect on CRISPR/Cas9-mediated chromosomal HDR. Overexpression of PML also inhibited both CRISPR HDR and I-SceI-induced HDR using a chromosomal reporter, and in an isoform-specific manner. However, the impact of PML overexpression on the chromosomal HDR reporter was dependent on the intranuclear chromosomal positioning of the reporter. Specifically, HDR at the TAP1 gene locus, which is associated with PML NBs, was reduced compared with a locus not associated with a PML NB; yet, HDR could be reduced at the non-PML NB-associated locus by PML overexpression. Thus, both loss and overexpression of PML isoforms can inhibit HDR, and proximity of a chromosomal break to a PML NB can impact HDR efficiency. |
| Databáze: | OpenAIRE |
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