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Note: A path to efficient gene editing Doi: 10.1038/s41591-018-0110-y Gene editing in human pluripotent stem cells (hPSCs) and iPSC -- the efficiency is very low compared to the other cell types. Kaykas and colleagues and Taipale and colleagues finding p53 antagonizing efficient genome editing using Cas9 in hPSC and immortalized human retinal pigment epithelial cells Editing gene that is silent in hPSCs -- > same result as editing one that active Single DSB induced by Cas9 in the genome of hPSC -- > reduces their survival Cas9 screens in transformed cells Copy number of gene target (rather than nature of individual genes) -- > drive phenotype of edited cells Thus -- clinical applications -- this could cause undesired side effects Kaykas and colleagues  Study transcriptional response of hPSC using gene editing with Cas9 Even one single strand break -- > increases expression of genes involved in programmed cell death Cas9-induced toxicity is mediated by p53 Reduced the le...

Note: Mechanisms of DNA double-strand break repair and their potential to induce chromosomal aberrations_2000 Doi: 10.1093/mutage/15.4.289 Theory of cancer formation Breakage and reunion theory Exchange theory Molecular theory Initial lesion -- chemical and physical-DNA damaging agents Artificially induced DSB IR Endonuclease Cellular (spontaneous) sources of DSB Can occur in any stage of cell cycle Briefly summarize the main causes of spontaneous DSB Topoisomerases Topo-I -- generates SSB Topo-II -- generate DSB Replication Prevalent source of DSB -- DNA replication Cell estimates to suffer ~ 10 DSB/cell cycle (estimate from spontaneous sister chromatid exchange) Meiosis V(D)J recombination Other recombinant process Transposable elements Fragile sites Extended micro- and minisatellite sequences -- potential source of DSB in mammalian genome At this time of writing (2000) -- the instability of this fragile site is not well-understood DSB as a results of excision repair Considering on t...

Note: Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin (doi: 10.1093/nar/gkn550) Three PI3KK can phosphorylate H2Ax; ATM, ATR and DNA-PK – each requires partner to recognize the break differently. MRN-ATM (heterochromatin region, S/G2-phase), ATRIP-ATR (stalled replication fork or bulk-damage entering to S-phase) and KU70/80-DNA-PK (occur throughout cell cycle). Scoring gH2Ax requires attention since it is not solely represented DSB. Remaining of gH2Ax foci does not mean the DSB has not been fixed. It may remain there to signal the HR to do more additional repair after seal with NHEJ, or the phosphatase system might be malfunction. The further condensation of the 30nm fiber as well as higher levels of chromatin condensation, which culminate with the 10 000-fold compaction of the stretched DNA fiber in the 700nm metaphase chromosomes, are less well understood, but are facilitated by the linker histone H1 and condensins. By ...

Note: DNA Damage Foci: Meaning and Significance (doi: 10.1002/em.21944) Basically, it can reflect DNA damage response but it cannot be solely related to DSB. (ionizing) radiation-induced foci (IRIF or RIF) or DNA repair foci. the histone variant H2AX which gets phosphorylated at its C-terminal Ser-139 residue by the DNA damage-activated kinases ATM, ATR, and DNA-PK, to form gH2AX. gH2AX then acts as a docking station for other DNA damage signaling factors such as MDC1 and 53BP1 which accumulate to form foci in a histone-modification-dependent manner. Foci can also be analysed using fluorescent protein fusion constructs, enabling foci formation and loss  to be monitored in live cells. As scoring is severely influenced by staining quality and imaging characteristics, it is good practice to include positive and negative reference samples which help confirm the validity and reproducibility of the results obtained in a particular experiment. Intensity-based approaches suc...

Note: Dynamic behavior of DNA topoisomerase IIbeta in response to DNA double-strand breaks (doi: 10.1038/s41598-018-28690-6) DNA topoisomerase II (Topo II) -plays important roles in various cellular processes, such as replication, transcription, and chromosome segregation. DNA topology problems may also occur during DNA repair, the possible involvement of Topo II in this process remains to be fully investigated. rapid recruitment of EGFP-tagged Topo IIβ to the DSB site. Topo IIβ is highly mobile in the nucleus. The Topo II catalytic inhibitors ICRF-187 and ICRF-193 reduced the Topo IIβ mobility and thereby prevented Topo IIβ recruitment to DSBs. Furthermore, Topo IIβ knockout cells exhibited increased sensitivity to bleomycin and decreased DSB repair mediated by homologous recombination (HR), implicating the role of Topo Iiβ in HR-mediated DSB repair. DNA topoisomerase II (Topo II) is an ATP-dependent enzyme that resolves DNA topological problems, such as superco...