Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.

Maintenance of human embryonic stem cells (hESCs) with stable genome is important for their future use in cell replacement therapy and disease modeling. Our understanding of the mechanisms maintaining genomic stability of hESC and our ability to modulate them is essential in preventing unwanted mutation accumulation during their in vitro cultivation. In this study, we show the DNA damage response mechanism in hESCs is composed of known, yet unlikely components. Clustered oxidative base damage is converted into DNA double-strand breaks (DSBs) by base excision repair (BER) and then quickly repaired by ligase (Lig)3-mediated end-joining (EJ). If there is further induction of clustered oxidative base damage by irradiation, then BER-mediated DSBs become essential in triggering the checkpoint response in hESCs. hESCs limit the mutagenic potential of Lig3-mediated EJ by DNA break end protection involving p53 binding protein 1 (53BP1), which results in fast and error-free microhomology-mediated repair and a low mutant frequency in hESCs. DSBs in hESCs are also repaired via homologous recombination (HR); however, DSB overload, together with massive end protection by 53BP1, triggers competition between error-free HR and mutagenic nonhomologous EJ.-Kohutova, A., Raska, J., Kruta, M., Seneklova, M., Barta, T., Fojtik, P., Jurakova, T., Walter, C. A., Hampl, A., Dvorak, P., Rotrekl, V. Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.

Dystrophin Deficiency Leads to Genomic Instability in Human Pluripotent Stem Cells via NO Synthase-Induced Oxidative Stress.

Recent data on Duchenne muscular dystrophy (DMD) show myocyte progenitor's involvement in the disease pathology often leading to the DMD patient's death. The molecular mechanism underlying stem cell impairment in DMD has not been described. We created dystrophin-deficient human pluripotent stem cell (hPSC) lines by reprogramming cells from two DMD patients, and also by introducing dystrophin mutation into human embryonic stem cells via CRISPR/Cas9. While dystrophin is expressed in healthy hPSC, its deficiency in DMD hPSC lines induces the release of reactive oxygen species (ROS) through dysregulated activity of all three isoforms of nitric oxide synthase (further abrev. as, NOS). NOS-induced ROS release leads to DNA damage and genomic instability in DMD hPSC. We were able to reduce both the ROS release as well as DNA damage to the level of wild-type hPSC by inhibiting NOS activity.