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

Study on the movement of lacO and spindle pole bodies in live yeast cells following HO expression and the formation of Rad52 foci after DNA damage. Investigation shows Rad52-GFP foci and SPBs move independently post-HO induction.

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Analysis of Double-Strand DNA Break in 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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