Localized movement and morphology of UBF1-positive nucleolar regions are changed by γ-irradiation in G2 phase of the cell cycle

Abstract

(2015) Localized movement and morphology of UBF1-positive nucleolar regions are changed by γ-irradiation in G2 phase of the cell cycle, Nucleus, 6:4, 301-313, DOI: 10.1080/19491034.2015 To link to this article: https://doi.org/10. 1080/19491034.2015 Abbreviations: 53BP1, p53-binding protein; ACT-D, actinomycin D; ATM, ataxia telangiectasia mutated; ATR, ATM and Rad3-related protein; AR, area ratio (area of nucleolus/area of nucleus); BrdU, 5-bromo-2 0 -deoxy-uridine; BER, base excision repair; CBs, Cajal bodies; DDR, DNA damage response; CPDs, cyclobutane pyrimidine dimers; DMEM, Dulbecco's modified Eagle's medium; DNA-PK, DNA-dependent protein kinase; DSBs, double-strand breaks; FA, foci area; NF, number of foci; FI, fluorescence intensity; FRAP, fluorescence recovery after photobleaching; GGR, global genome repair; GFP, green fluorescence protein; HP1, heterochromatin protein 1; HR, homologous recombination; HRR, homologous recombination repair; iMEFs, immortalized mouse embryonic fibroblasts; LR, local radius; NBs, nuclear bodies; NER, nucleotide excision repair; NHEJ, non-homologous end-joining; nA, nucleolus area; NA, nucleus area; PBS, phosphate-buffered saline; P2A, compactness; PCR, polymerase chain reaction; PML, promyelocytic leukemia bodies; rDNA, ribosomal DNA; RNA Pol II, RNA polymerase II; ROI, region of interest; RPA, replicationrelated protein A; SEM, standard error of the mean; SDS, sodium dodecyl sulfate; TCR, transcription-coupled NER; UBF1, upstream binding factor 1; UV, ultraviolet.The nucleolus is a well-organized site of ribosomal gene transcription. Moreover, many DNA repair pathway proteins, including ATM, ATR kinases, MRE11, PARP1 and Ku70/80, localize to the nucleolus (Moore et al., 2011). We analyzed the consequences of DNA damage in nucleoli following ultraviolet A (UVA), C (UVC), or g-irradiation in order to test whether and how radiation-mediated genome injury affects local motion and morphology of nucleoli. Because exposure to radiation sources can induce changes in the pattern of UBF1-positive nucleolar regions, we visualized nucleoli in living cells by GFP-UBF1 expression for subsequent morphological analyses and local motion studies. UVA radiation, but not 5 Gy of g-rays, induced apoptosis as analyzed by an advanced computational method. In non-apoptotic cells, we observed that g-radiation caused nucleolar re-positioning over time and changed several morphological parameters, including the size of the nucleolus and the area of individual UBF1-positive foci. Radiation-induced nucleoli rearrangement was observed particularly in G2 phase of the cell cycle, indicating repair of ribosomal genes in G2 phase and implying that nucleoli are less stable, thus sensitive to radiation, in G2 phase.

Distinct kinetics of DNA repair protein accumulation at DNA lesions and cell cycle‐dependent formation of γH2AX‐ and NBS1‐positive repair foci

“…FRAP analyses were then performed 90–120 min after γ‐irradiation. Since UVA but not 5Gy of γ‐rays induced apoptotic fragmentation of lamin B, we concluded that γ‐irradiation induced DNA repair‐related foci but not apoptotic processes (Sorokin et al., ). Similar irradiation conditions we used here.…”

Nucleolar Reorganization Upon Site-Specific Double-Strand Break Induction

“…This response of nucleolus to cellular stress has been known for decades. 17,40,41 Nucleolar reorganization is often studied by ActD exposure for a short period of time. This produces NCs, most likely as a consequence of transcriptional silencing.…”

A role of the 53BP1 protein in genome protection: structural and functional characteristics of 53BP1-dependent DNA repair

“…Together, FRAP, time-lapse microscopy, and single-particle tracking analyses represent a valuable biophysical approach for studying the dynamic properties of DNA repair proteins [60,63–66]. These experimental tools, which are useful for live cell studies, are essential for understanding the functional properties and hierarchical binding of repair proteins to damaged DNA.…”