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

Abstract

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.

Imaging chromatin nanostructure with binding-activated localization microscopy based on DNA structure fluctuations

“…This prompted us to conclude that the unbound YOYO-1 was washed away, but some YOYO-1 stayed bound to the remaining dsDNA. This is in line with our recent finding that even exposing DNA in fixed cells to pH <3, denaturation is not complete ( 46 ). Lastly, similar results have been obtained for other cyanine dyes in the presence of low pH, primary thiols ( Supplementary Figure S2 ), and also in buffers of an increased ionic strength ( Supplementary Figure S3 , discussed in Supplementary Note 1 ).…”

Low level phosphorylation of histone H2AX on serine 139 (γH2AX) is not associated with DNA double-strand breaks

“…We, as well as others, have shown that EdU is a sensitive marker of replication, with no detectable nonspecific staining or background [33]. Our microscopy system is capable of detecting DNA replication with high sensitivity, therefore the rate of false negative identification of S-phase cells is very low if any.…”

Induction of apoptosis and ferroptosis by a tumor suppressing magnetic field through ROS-mediated DNA damage

“…The proliferation rate was the calculated ratio of the number of cells with nuclei fully incorporated with EdU against the total number of cell nuclei visualized by Hoechst 33342. Meanwhile, loci of EdU partial incorporation in the nuclei indicate unscheduled DNA synthesis, i.e., occurrence of newly synthesized DNA for damage repair [59], was also counted.…”