PMC:3583298 / 12650-15932 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/3583298","sourcedb":"PMC","sourceid":"3583298","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3583298","text":"YAP over-expression affects DNA repair and causes genomic instability\nDefects in cell cycle checkpoints might result in genomic instability. We asked at which level YAP was affecting the DDR. Breaks in DNA are marked by the formation of protein complexes termed IR-induced foci (IRIF), which can be identified when analyzed by immunostaining for proteins comprising these complexes. To determine whether YAP-infected cells had reduced DNA damage, we carried out immunostaining for p53 binding protein 1 (53BP1), a key mediator of the DDR often used as a marker of the IRIF. At 3 hours post-irradiation, GFP- and YAP-infected CGNPs had similar numbers of foci per cell (data not shown). However, at 9 and 24 hours post-irradiation, YAP-infected CGNPs had a significantly reduced number of cells containing foci as well as fewer foci per cell (Figure 3A). These findings would suggest that YAP expression either enables cells to repair DNA more rapidly, causing foci to resolve earlier, or that YAP expression inhibits DNA repair pathways, resulting in dismantling of the DNA repair complexes.\nTo distinguish between these two possibilities, we carried out comet assays to detect broken DNA, in irradiated GFP- or YAP-infected CGNPs. In the comet assay, cells are immersed in an agarose matrix extended on a microscope slide, and treated with a lysis solution. Nuclear DNA is unwound under alkaline conditions and fragments resulting from strand breaks can migrate away from the nuclei under an electric field, forming a “comet tail” when stained. We quantified the amount of DNA damage by measuring tail lengths. As shown in Figure 3B, comet tails can be detected in GFP-infected CGNPs at 9 hours post-irradiation, but to a lesser extent at 24 hours post-irradiation, indicating that most of the damaged DNA is repaired at that point.\nIn contrast, at 9 hours after irradiation, YAP-infected cells had strikingly pronounced comet tails. Comet tails persisted in YAP-infected CGNPs 24 hours after irradiation, a time point at which we can detect proliferation in these cells (Figure 2). In order to analyze whether the DNA repair defect leads to chromosomal alterations, we performed metaphase spreads on GFP- or YAP-infected CGNPs 24 hours after irradiation. As shown in Figure 3C, YAP infection is associated with an increase in the number of cells with persistent DNA breaks. We also observed that YAP-transduced Pzp53med cells (Berman et al 2002) showed a similar loss of irradiation-induced foci and increased number of cells with DNA breaks after irradiation (Supplementary Figure 3A, B). These observations indicate that YAP expression enables cells to proliferate with unrepaired DNA, a condition which can lead to genomic instability, a hallmark of cancer cells. To address whether the effect of YAP was specific for radiation-induced DNA damage, we treated GFP- and YAP-infected cells with the antibiotic Bleomycin, which causes double strand breaks in DNA. Similar to what we observed in irradiated cells, 9 and 24 hours after treatment there were significantly fewer cells that contained foci among those over-expressing YAP, which indicates that the role of YAP promoting focus dis-assembly is not specific to radiation-induced damage in the DNA (Supplementary Figure 3C).","divisions":[{"label":"title","span":{"begin":0,"end":69}},{"label":"p","span":{"begin":70,"end":1091}},{"label":"p","span":{"begin":1092,"end":1833}}],"tracks":[{"project":"2_test","denotations":[{"id":"21874045-12202832-79138690","span":{"begin":2442,"end":2446},"obj":"12202832"},{"id":"21874045-12202832-79138690","span":{"begin":2442,"end":2446},"obj":"12202832"}],"attributes":[{"subj":"21874045-12202832-79138690","pred":"source","obj":"2_test"},{"subj":"21874045-12202832-79138690","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#939dec","default":true}]}]}}