Analysis of DNA double-strand break response and chromatin structure in mitosis using laser microirradiation
| Autor: | Kyoko Yokomori, Michael W. Berns, Adria J. Sherman, You Zhongsheng, Lih-Huei L. Liaw, Tao Wu, Christopher S. Lee, Veronica Gomez-Godinez |
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| Rok vydání: | 2010 |
| Předmět: |
Male
nucleolar organizer DNA damage mammalian-cells Mitosis Biology ubiquitin ligase DNA-binding protein Chromosomes Cell Line law.invention 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Potoroidae law Microscopy Genetics Animals DNA Breaks Double-Stranded Microscopy Phase-Contrast DAPI Ku Autoantigen potorous-tridactylis 030304 developmental biology 0303 health sciences damage response Ubiquitin optical tweezers Lasers Nuclear Proteins Life Sciences histone h2ax phosphorylation Antigens Nuclear Laser Chromatin Cell biology DNA-Binding Proteins Radiography nuclear foci chemistry mre11 complex Methods Online Female living cells 030217 neurology & neurosurgery DNA |
| Zdroj: | Gomez-Godinez, Veronica; Wu, Tao; Sherman, Adria J.; Lee, Christopher S.; Liaw, Lih-Huei; Zhongsheng, You; et al.(2010). Analysis of DNA double-strand break response and chromatin structure in mitosis using laser microirradiation. Nucleic Acids Research, 38(22), e202-e202. UC Irvine: Retrieved from: http://www.escholarship.org/uc/item/0rd9p8vn Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| Popis: | In this study the femtosecond near-IR and nanosecond green lasers are used to induce alterations in mitotic chromosomes. The subsequent double-strand break responses are studied. We show that both lasers are capable of creating comparable chromosomal alterations and that a phase paling observed within 1-2 s of laser exposure is associated with an alteration of chromatin as confirmed by serial section electron microscopy, DAPI, γH2AX and phospho-H3 staining. Additionally, the accumulation of dark material observed using phase contrast light microscopy (indicative of a change in refractive index of the chromatin) ∼ 34 s post-laser exposure corresponds spatially to the accumulation of Nbs1, Ku and ubiquitin. This study demonstrates that chromosomes selectively altered in mitosis initiate the DNA damage response within 30 s and that the accumulation of proteins are visually represented by phase-dark material at the irradiation site, allowing us to determine the fate of the damage as cells enter G1. These results occur with two widely different laser systems, making this approach to study DNA damage responses in the mitotic phase generally available to many different labs. Additionally, we present a summary of most of the published laser studies on chromosomes in order to provide a general guide of the lasers and operating parameters used by other laboratories. |
| Databáze: | OpenAIRE |
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