Subnuclear localization, rates and effectiveness of UVC-induced unscheduled DNA synthesis visualized by fluorescence widefield, confocal and super-resolution microscopy

Unscheduled DNA synthesis (UDS) is the final stage of the process of repair of DNA lesions induced by UVC. We detected UDS using a DNA precursor, 5-ethynyl-2'-deoxyuridine (EdU). Using wide-field, confocal and super-resolution fluorescence microscopy and normal human fibroblasts, derived from h...

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Vydané v:Cell cycle (Georgetown, Tex.) Ročník 15; číslo 8; s. 1156 - 1167
Hlavní autori: Pierzyńska-Mach, Agnieszka, Szczurek, Aleksander, Cella Zanacchi, Francesca, Pennacchietti, Francesca, Drukała, Justyna, Diaspro, Alberto, Cremer, Christoph, Darzynkiewicz, Zbigniew, Dobrucki, Jurek W.
Médium: Journal Article
Jazyk:English
Vydavateľské údaje: United States Taylor & Francis 01.01.2016
Predmet:
Bromodeoxyuridine - metabolism
Cell Nucleus - metabolism
Cell Nucleus - radiation effects
Cells, Cultured
Deoxyuridine - analogs & derivatives
Deoxyuridine - metabolism
DNA - biosynthesis
Dose-Response Relationship, Radiation
Fibroblasts - metabolism
Fibroblasts - radiation effects
Fluorescence
Humans
Microscopy, Confocal - methods
Nucleic Acid Denaturation - radiation effects
Subcellular Fractions - metabolism
Subcellular Fractions - radiation effects
Time Factors
Ultraviolet Rays
cytometry
unscheduled DNA synthesis
UDS
DNA damage
DNA repair
single molecule localization microscopy
super-resolution microscopy
ultraviolet light
confocal microscopy
fluorescence
EdU
dSTORM
NER
BrdU
nucleotide excision repair
ISSN:1538-4101, 1551-4005, 1551-4005
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Shrnutí:Unscheduled DNA synthesis (UDS) is the final stage of the process of repair of DNA lesions induced by UVC. We detected UDS using a DNA precursor, 5-ethynyl-2'-deoxyuridine (EdU). Using wide-field, confocal and super-resolution fluorescence microscopy and normal human fibroblasts, derived from healthy subjects, we demonstrate that the sub-nuclear pattern of UDS detected via incorporation of EdU is different from that when BrdU is used as DNA precursor. EdU incorporation occurs evenly throughout chromatin, as opposed to just a few small and large repair foci detected by BrdU. We attribute this difference to the fact that BrdU antibody is of much larger size than EdU, and its accessibility to the incorporated precursor requires the presence of denatured sections of DNA. It appears that under the standard conditions of immunocytochemical detection of BrdU only fragments of DNA of various length are being denatured. We argue that, compared with BrdU, the UDS pattern visualized by EdU constitutes a more faithful representation of sub-nuclear distribution of the final stage of nucleotide excision repair induced by UVC. Using the optimized integrated EdU detection procedure we also measured the relative amount of the DNA precursor incorporated by cells during UDS following exposure to various doses of UVC. Also described is the high degree of heterogeneity in terms of the UVC-induced EdU incorporation per cell, presumably reflecting various DNA repair efficiencies or differences in the level of endogenous dT competing with EdU within a population of normal human fibroblasts.
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Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/kccy.
ISSN:1538-4101
1551-4005
1551-4005
DOI:10.1080/15384101.2016.1158377