Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.

Science 13 July 2007:
Vol. 317. no. 5835, pp. 242 - 245
DOI: 10.1126/science.1140649
Prev | Table of Contents | Next

Reports

Postreplicative Formation of Cohesion Is Required for Repair and Induced by a Single DNA Break

Lena Ström1, Charlotte Karlsson1, Hanna Betts Lindroos1, Sara Wedahl1, Yuki Katou2, Katsuhiko Shirahige2 and Camilla Sjögren1*

1 Department of Cell and Molecular Biology, Karolinska Institute, 171 77 Stockholm, Sweden.
2 Gene Research Centre, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, 226-8501 Yokohama, Japan.


Figure 1 Fig. 1. Mre11, Mec1, Tel1, and {gamma}H2A, but not Rad9 and Rad52, are required for {gamma}-ray–induced cohesion. (A to H) Chromatid separation at URA3 on chr. V in G2/M-arrested cells after {gamma}-irradiation and destruction of S phase–established cohesion, as described in fig. S1A. (A) Chromatid separation in Cdc20-depleted G2/M-arrested smc1-259, GAL:SMC1-13MYC, MET:CDC20 cells (CB496). (B to H) Chromatid separation in nocodazole G2/M-arrested (B) smc1-259, GAL:SMC1-13MYC cells (CB469), combined with (C) mre11{Delta} (CB478), (D) mec1{Delta} (CB784), (E) tel1{Delta} (CB693), (F) hta1-S129stop, hta2-129stop (CB742), (G) rad9{Delta} (CB696), or (H) rad52{Delta} (CB571). Gal, galactose addition; IR, irradiation. [View Larger Version of this Image (28K GIF file)]
 

Figure 2 Fig. 2. A single DSB on chr. III leads to establishment of cohesion on chr. V. MCD1UNCL was induced in G/M-arrested cells, with or without a concomitant DSB on chr. III. Cells were thereafter released into the next cell cycle under non-inducing conditions (fig. S1C). Chromatid separation at URA3 on chr. V, thepercentageof Pds1-positive cells, and chr. III breakage were determined. (A) Sister-chromatid separation in GAL:MCD1UNCL, PDS1-18MYCcells without (CB699) or with GAL:HO (CB507). (B) Pds1-positive cells in (A). (C) Southern blot of chr. III isolated from cells examined in (A). (D) Sister-chromatid separation in GAL:MCD1 (CB699), GAL:MCD1 GAL:HO (CB507), GAL:HO (CB524), or MCD1UNCL GAL:HO mat{Delta} (CB586). (E) Analysis of chr. III in cells examined in (D). [View Larger Version of this Image (35K GIF file)]
 

Figure 3 Fig. 3. Genome-wide cohesion depends both on the DNA damage response and on proteins regulating cohesin function. (A and B) Chr. V chromatid separation in G2/M-arrested cells after removal of S phase–established cohesion, in the absence or presence of a DSB on chr. III, as described in fig. S1B. (A) Chromatid separation in smc1-259, GAL:HO (CB583), and smc1-259 GAL:SMC1-13MYC GAL:HO (CB479). (B) Chromatid separation in smc1-259, GAL: SMC1-13MYC, GAL:HO cells (CB479), combined with tel1{Delta} (CB815), mec1{Delta} (CB753), hta1-S129stop, hta2-129stop (CB740), or rad9{Delta} (CB813). DSB formation on chr. III is shown in fig. S3A (C to E) Chromatid separation of chr. V in cells containing GAL:MCD1UNCL and GAL:HO. The experiments were performed as in Fig. 2 and fig. S1C, with the exception that the temperature was up-shifted 30 min after the addition of galactose. (C) Wild type (CB507) and scc2-4 (CB573), (D) wild type and smc6-56 (CB537), and (E) wild type and eco1-1 (CB732). (F) Chip-on-chip analysis of Scc2 localizationon chr. III in the absence (–DSB) or presence (+DSB) of a DSB at the MAT locus. Arrow indicates a DSB. CENIII, chr. III centromere. [View Larger Version of this Image (33K GIF file)]
 

Figure 4 Fig. 4. Postreplicative function of Eco1 is required for DSB repair but dispensable for chromosome segregation. (A and B) DNA repair and sister-chromatid separation at URA3 in G2/M-arrested WT (CB167) and eco1-1 (CB720) cells. After arrest in G2/M at 21°C, half of the cultures were transferred to 32°C for 30 min, and then all cells were treated with 200 grays of {gamma}-irradiation (IR) (1 gray = 100 rads). At indicated time points and temperatures, samples were withdrawn for analyses of DNA repair by pulsed-field gel electrophoresis (PFGE) and sister-chromatid separation (29). (Left) Southern blots of the PFGE gel with the use of a radioactive probe detecting chr. XVI and a loading control. (Middle) Quantification of chr. XVI signals normalized to the control. (Right) Chromatid separation. (C) Chromosome segregation in the absence (–) and presence (+) of genome-wide damage-induced cohesion. G2/M-arrested WT (CB524) or temperature-sensitive eco1-1 (CB755) cells were treated such that damage-induced cohesion was inhibited in eco1-1 cells (29). A DSB on chr. III was induced in half of the cultures. After release into a G1 arrest, the percentage of cells with a single chr. V, reflecting correct chromosome segregation, was determined. [View Larger Version of this Image (34K GIF file)]
 

To Advertise     Find Products

Science. ISSN 0036-8075 (print), 1095-9203 (online)