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Analysis of a Double-Strand DNA Break in living S. cerevisiae Cells

Analysis of a Double-Strand DNA Break in living S. cerevisiae Cells. Leana M. Topper*, L. Kevin Lewis # , Kerry S. Bloom*, and Michael A. Resnick #. *Department of Biology, University of North Carolina at Chapel Hill; # National Institute of Environmental Health Sciences,

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Analysis of a Double-Strand DNA Break in living S. cerevisiae Cells

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  1. Analysis of a Double-Strand DNA Break in living S. cerevisiae Cells Leana M. Topper*, L. Kevin Lewis#, Kerry S. Bloom*, and Michael A. Resnick# *Department of Biology, University of North Carolina at Chapel Hill; #National Institute of Environmental Health Sciences, NIH Research Triangle Park

  2. Chromosome III MAT CEN ~85 kb LacO array ~500 bp

  3. MAT LacO KpnI KpnI probe 2.5 kb No pGalHOT W/ pGalHOT 0 0.5 2 3 4 1 0 0.5 2 3 4 1 h post gal 8 kb KpnI fragment HO cut fragment

  4. Live cell after HO induction 6 min 7 min 2 min 13 min 8 min 10 min 15 min

  5. Movement of Spindle Pole Bodies and lacO in live cells following HO expression Average film time: 13 min No bud: 13 Small-budded cells: 8 Large-budded cells: 29 Average spindle length = 1.69 ±0.32mm Range = 1.05-2.44 mm

  6. Population analysis of lacO and SPBs after induction of HO

  7. Deletion of Rad52 does not affect LacO movement 3 min 6 min 0 min 11 min 13 min 18 min

  8. Formation of Rad52 foci following DNA damage

  9. Formation of Rad52-GFP foci after induction of HO

  10. Rad52-GFP foci and Spindle Pole Bodies Move Independently 1 min 5 min 7 min 17 min 8 min 12 min 18 min

  11. Rad52-CFP and LacO spots do not colocalize

  12. lacO spc29 following HO induction

  13. Rad52 delete following HO induction

  14. Rad52-CFP, LacO-GFP upon HO induction

  15. Rad52 (green) spc 29 (in red)

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