Post on 10-Jan-2016
description
Analysis of a Double-Strand DNA Break in living S. cerevisiae Cells
Leana M. Topper Kerry S. Bloom
Department of Biology, University of North Carolina at Chapel Hill;
Chromosome III
CEN MAT
~85 kb
LacO array
~500 bp
8 kb KpnI fragment
HO cut fragment
KpnI KpnILacO MATprobe
2.5 kb
No pGalHOT W/ pGalHOT
0 0.5 1 2 3 4 0 0.5 1 2 3 4 h post gal
Live cell after HO induction
2:00 6:00 7:00
15:008:00 10:00 13:00
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: 29Average spindle length = 1.69 ±0.32
mRange = 1.05-2.44 m
Time post gal addition No bud Small bud Large bud
0 min 8 9 12
30 min 3 3 10
1 h 2 8 9
2 h 3 9 8
3 h 8 5 6
4 h 10 6 14
Total 26 31 47
Population analysis of lacO and SPBs after induction of HO
Deletion of Rad52 does not affect LacO movement
0:00 2:00 6:00
9:00 15:00 20:00 21:00
Formation of Rad52 foci following DNA damage
Time post gal
No bud
fociSmall bud
fociLarge bud
fociDivided nucleus
0 h 36 0 133
(23%)12
1
(8.3%)9
1-2 h 84 0 23 0 454
(8.8%)21
2-3 h 75 0 222
(9.0%)46
9
(19.6%)13
3-4 h 1042
(1.9%)18 4 42
8
(19.0%)21
Formation of Rad52-GFP foci after induction of HO
Rad52-GFP foci and Spindle Pole Bodies Move Independently
1:00 5:00
8:00 12:00 17:00 18:00
7:00
Rad52-CFP and LacO spots do not colocalize
3:00 8:006:00
9:00 11:00 14:00 16:00