Activation of new replication foci under conditions of replication stress

Abstract

Abbreviations: Cpt, camptothecin; Tpt, topotecan; EdU, 5-ethynyl-2 0 -deoxyuridine; BrdU, 5-bromo-2 0 -deoxyuridine; PCNA, proliferating cell nuclear antigen; EGFP, Enhanced Green Fluorescent Protein; gH2AX, histone H2AX phosphorylated on Ser139.DNA damage, binding of drugs to DNA or a shortage of nucleotides can decrease the rate or completely halt the progress of replication forks. Although the global rate of replication decreases, mammalian cells can respond to replication stress by activating new replication origins. We demonstrate that a moderate level of stress induced by inhibitors of topoisomerase I, commencing in early, mid or late S-phase, induces activation of new sites of replication located within or in the immediate vicinity of the original replication factories; only in early S some of these new sites are also activated at a distance greater than 300 nm. Under high stress levels very few new replication sites are activated; such sites are located within the original replication regions. There is a large variation in cellular response to stress -while in some cells the number of replication sites increases even threefold, it decreases almost twofold in other cells. Replication stress results in a loss of PCNA from replication factories and a twofold increase in nuclear volume. These observations suggest that activation of new replication origins from the pool of dormant origins within replication cluster under conditions of mild stress is generally restricted to the original replication clusters (factories) active at a time of stress initiation, while activation of distant origins and new replication factories is suppressed.

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

“…Maximum z-projection images and 3D reconstructions, shown in Figure 3, convey the information about the overall density of foci of the three types, while the central cross-sections, shown in Supplementary Figure 1, reveal more clearly the characteristic patterns of replication foci in subsequent sub-stages of S-phase. The actual numbers of foci are given in Figure 4 and Supplementary Figure 3 (for all substages of S-phase; the particular substages were identified based on a characteristic pattern of spatial location of DNA replication sites, as described before [25, 26]). There was a great variation in sizes of the immunostained phosphorylated regions of chromatin, and a wide range of fluorescence brightness, a parameter, which is proportional to the amount of γH2AX in each focus (Supplementary Figure 2).…”

“…A549 human lung adenocarcinoma cells were obtained from ATCC and maintained as described previously [26]. Exposures to the drugs were commenced 24–48 h after seeding, when cells were in the exponential phase of growth and reached approximately 70% confluency.…”

“…Determination of the parameters was unbiased by drug treatment or the classification of the sub-stage of the S phase of the cell cycle. Estimation of the foci volume was based on an iterative flooding of the maxima in 3D space as described previously in [26]. Classification of γH2AX foci into two classes was based on foci volume and mean fluorescence intensity in raw images, prior to deconvolution.…”

Murine polyomavirus DNA transitions through spatially distinct nuclear replication subdomains during infection

“…localization of EdU signal at VRCs, which co-localizes with vDNA labeled by FISH [8]. Cells were excluded from analysis if they exhibited signs of host DNA replication, such as EdU signal along the nuclear periphery or in perinucleolar space [88][89][90].…”

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

“…[32][33][34] We have previously noticed significant increase of the nuclear volume following DNA damage (dsDNA breaks) induced by topotecan. 35 This increase may be caused by global chromatin decondensation observed during DNA repair. [35][36][37] Here, in the case of UV-induced damage, no significant increase of nuclear volumes was observed within 6 hours of UV insult (Fig 5A,C), suggesting that large scale chromatin decondensation may not be required for NER at that time point after the UV exposure.…”