Abstract
Cell-cycle progression can be arrested by ionizing radiation-induced DNA double-strand breaks (DSBs). Although DSBs are patched by DSB repair systems, which comprise proteins such as p53-binding protein 1 (53BP1), the relationship between DSB repair progression and cell-cycle status in living cells is unclear. The probe FUCCI (fluorescent ubiquitination-based cell-cycle indicator) was previously developed for visualizing cell-cycle status. Here, we established novel live-imaging probes based on custom-designed plasmids designated “Focicles” harboring a tricistronic compartment encoding distinct fluorescent proteins ligated to the murine 53BP1 foci-forming region (FFR) and two cell-cycle indicators that are known components of FUCCI (hCdt1 and hGmnn). We used CRISPR/Cas9-mediated genome editing to obtain Focicle knock-in cell lines in NIH3T3 cells, which were subject to X-ray irradiation that induced comparable numbers of Focicle and endogenous-53BP1 foci. In addition, the Focicle probes enabled the kinetic analysis of both DSB repair and cell-cycle arrest/progression after irradiation, demonstrating that the Focicle knock-in cells progressed to cell division after DNA damage elimination. These newly developed probes can help to gain a better understanding of the dynamics of DSB repair and cell-cycle control to in turn guide cancer treatment development and cancer-risk assessments.
Highlights
Visualization of intracellular molecules through fluorescent live imaging is a powerful technique for uncovering the biological dynamics of cells[1,2,3]
All inserts encode fusion proteins composed of the two cell-cycle indicators and m53BP1FFR (Supplementary Table S1 and Supplementary Methods) each connected to an fluorescent proteins (FPs)
The sequence of m53BP1FFR was obtained from cDNA of mouse colonic cells (Supplementary Fig. S2)
Summary
Visualization of intracellular molecules through fluorescent live imaging is a powerful technique for uncovering the biological dynamics of cells[1,2,3]. We developed a new live-imaging system using custom-designed plasmids named “Focicle” (“foci” + “cell cycle”), which harbored a tricistronic cassette encoding the FFR of mouse 53BP1 (m53BP1FFR) and two cell-cycle indicators (hCdt[1] and hGmnn) fused to distinct FPs. We established Focicle-knock-in cell lines in which the constructs were inserted at the ROSA26 locus, a well-known mouse safe-harbor site[14], using CRISPR/Cas9-mediated genome editing. We evaluated the progression of DSB repair and the cell-cycle status in Focicle knock-in cells after exposure to ionizing radiation.
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