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