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Figures
- Fig. 1.
Construction of fluorescently tagged strains. (A) Frogging of fluorescently tagged strains on various genotoxic drugs. Drug concentrations are as follows: CPT, 0.0125 mM; HU, 7.5 mM; MMS, 0.012%; Phleo, 1.5 mU. (B) Fluorescently tagged strains Pku70-Citrine::hph and Mre11-mCherry::hph both with and without genotoxic drugs. For clarity Mre11-mCherry is shown in false color as magenta. Arrows show representative foci for Pku70 and generalized areas of increased fluorescence for Mre11. (C) Pku-Citrine foci were counted by hand and significance testing was done using a Mann–Whitney significance test. Six replicates were used per drug tested.
- Fig. 2.
Pku70-Citrine colocalization with DNA repair proteins. (A) These images depict colocalization of Pku70-Citrine with previously reported HR repair proteins Rad52-mCherry and RPA-CFP under four commonly used genotoxic drugs. For clarity Rad52-mCherry is shown in false color magenta, Pku70-Citrine is being shown in false color as green. In the composite image overlapping foci appear as white. Arrows show examples of easily visible colocalization. (B) Percent of nuclei with either 1 or ≥2 Pku70-Citrine foci. (C) Percent of Pku70 foci that colocalize with either a Rad52 foci or a RPA foci. (D) Percent of either Rad52 or RPA foci that have a corresponding colocalizing Pku70 foci.
- Fig. 3.
Colocalization of Mre11-mCherry, Rad52-YFP, RPA-CFP and Pku70-Citrine. (A) Cells were treated in 0.45 mM MMS for 4 h at 32°C. Mre11-mCherry is shown in false color as magenta and Rad52-YFP is shown in green for clarity. (B) Time-lapse microscopy of Mre11-mCherry and Pku70-Citrine. Cells were treated in 0.45 mM MMS and time-lapses were kept 28°C. Timepoints designate time since drug addition.
- Fig. 4.
Pku70 localization in a dynamic timecourse of MMS treatment. (A) Fluorescent time lapse images of Pku70-Citrine colocalizing with Rad52-mcherry. For clarity Rad52-mCherry is shown in magenta and Pku-Citrine is shown in green. Imaging was started at 80 min post addition of 0.45 mM MMS and cells were imaged at 28°C. Time-course images were taken every 20 min. (B) Persistence time of Rad52-mCherry (n=205) and Pku70-Citrine (n=195). (C) Appearance and disappearance times for Pku70-Citrine and Rad52-YFP in individual mononucleate cells. T=0 first timepoint after completion of cytokinesis. Horizontal density shows higher quantity of foci appearing or disappearing at that timepoint. (n=35 cells).
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