Volume 22, Issue 6, Pages 755-767 (June 2006) Chromosomal Association of the Smc5/6 Complex Reveals that It Functions in Differently Regulated Pathways  Hanna Betts Lindroos, Lena Ström, Takehiko Itoh, Yuki Katou, Katsuhiko Shirahige, Camilla Sjögren  Molecular Cell  Volume 22, Issue 6, Pages 755-767 (June 2006) DOI: 10.1016/j.molcel.2006.05.014 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Smc6 and Nse1 Localization Is Concentrated to the Centromeric Region of ChrVI in Unchallenged, G2/M-Arrested Cells (A and B) Cells with Flag-tagged (A) Smc6 (CB313) or (B) Nse1 (CB354) were arrested in G1, S, or G2/M at 23°C. Upon arrest in respective cell cycle phase, samples for fixation and subsequent analysis by ChIP on chip were collected and analyzed on ChrVI arrays (Experimental Procedures). S phase cells were incubated for 2.5 hr in HU containing media after release from G1 arrest. Blue horizontal lines indicate the open reading frames, and orange peaks indicate the significant binding of the proteins to the chromosome. CEN denotes the position of the centromere, and the red lines and numbers indicate the positions of autonomously replication origins (ARS). The horizontal lines indicate log 1 of the signal strength, and the vertical scale bar indicates the chromosomal coordinates in kb. Molecular Cell 2006 22, 755-767DOI: (10.1016/j.molcel.2006.05.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Smc6 Associates with the Genome after Replication and Binds Specifically to All Centromeres and to the Arms of Long Chromosomes (A) Localization of Smc6 on a representative long chromosome (IV, left arm) and to the area downstream of the rDNA repeats on ChrXII in unchallenged cells arrested in G2/M. Smc6-Flag expressing (CB499) cells were arrested in G2/M when samples for ChIP on chip were collected and hybridized to whole genome arrays (Experimental Procedures). (B) Correlation between chromosome length and the number of Smc6 interaction sites per kb on each chromosome. Forty kilobases around each centromere are excluded from the analysis. Abbreviation: cc, correlation coefficient. (C) Comparison of the chromosomal localization of Smc6 and Scc1. w/w indicates peaks in intergenic regions between two genes on the Watson strand. w/c, c/w, and c/c indicate peaks in intergenic regions between genes on the Watson/Crick, Crick/Watson and Crick/Crick strands, respectively. Orf denotes the number of peaks within an open reading frame. The circle diagram visualizes this analysis. (D) Smc6 localization in S phase to the same chromosomal regions as in (A). Smc6-Flag expressing (CB499) cells were arrested in S phase and incubated as in Figure 1. Samples for ChIP on chip were collected, processed, and analyzed as in (A). Annotations as in Figure 1. Molecular Cell 2006 22, 755-767DOI: (10.1016/j.molcel.2006.05.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Lack of Scc2 or Scc1 during Replication Influences the Localization of Smc6 to Chromosomes in G2/M (A) Smc6-Flag expressing (CB499) cells were arrested in G1, and then the temperature was raised to 37°C for 30 min during maintained G1 arrest. Subsequently, cells were released into benomyl containing media at restrictive temperature, and samples for whole genome analysis by ChIP on chip were collected after 2.5 hr, when all cells had reached G2/M. The left and right panels show the first 400 kb of ChrV and the 400 kb downstream of the rDNA repeats on ChrXII, respectively. (B and C) Temperature-sensitive ssc2-4 (CB425) or scc1-73 (CB617) cells expressing Smc6-Flag were arrested and released as in (A) and passed through replication at restrictive temperature into a benomyl-activated G2/M arrest when samples for whole genome ChIP on chip analysis were collected. Annotations as in Figure 1. Molecular Cell 2006 22, 755-767DOI: (10.1016/j.molcel.2006.05.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Smc6 Associates with the Region Surrounding a DSB 30 Min after Initiation of Break Induction and Is Required for Efficient Repair of γ-Ray-Induced DNA Damage (A and B) Cells expressing Flag-tagged Smc6 (CB313) or Nse1 (CB354), both containing GAL:HO and a recognition site for HO inserted 206.5 kb from the left telomere of ChrVI, were arrested in G2/M. Two percent galactose for activation of GAL:HO was then added to the cultures. Samples for ChIP on chip were collected at the indicated time points and analyzed on ChrVI arrays. An arrow marks the location of the HO-induced DSB, other annotations as in Figure 1. (C) DNA repair in SMC6 and temperature-sensitive smc6-56 cells. SMC6 (CB67) or smc6-56 (CB309) cells were arrested in G2/M by addition of nocodazole at 21°C. Upon arrest in G2/M, the temperature was raised to 35°C for 30 min to destroy smc6-56 function. Cells were then irradiated with 250 Gy. Samples for preparation of chromosomes were collected before, just after irradiation, and at indicated time points during a recovery period during which the cells were kept in G2/M arrest by the presence of nocodazole. Chromosomes were then separated by PFGE and analyzed by Southern blot using a ChrXVI radioactive probe detecting both the sample ChrXVI and a loading control. (D) Quantification of the results from (C). The ChrXVI signals obtained by Southern blot were quantified and normalized to those of the loading control. The value obtained from the sample collected before irradiation was set to 100% arbitrary units. Molecular Cell 2006 22, 755-767DOI: (10.1016/j.molcel.2006.05.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Smc6 Is Recruited to Collapsed Replication Forks (A and B) Wild-type (CB499) or rad53▵ (CB453) cells expressing Smc6-Flag were arrested in S phase by 1 hr incubation in HU containing media after release from G1 arrest. Samples for ChIP on chip were then collected and analyzed on ChrVI arrays. Annotations as in Figure 1. Molecular Cell 2006 22, 755-767DOI: (10.1016/j.molcel.2006.05.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Localization of Smc6 to an HO-Induced DSB Requires Mre11, but Not Mec1, Rad53, or Scc2 (A) Wild-type (CB499), mre11▵ (CB455), mec1▵ (CB461), or rad53▵ (CB470) cells expressing Smc6-Flag, all containing GAL:HO and a HO cut site at 206.5 on ChrVI, were arrested in G2/M. The HO-induced DSB was then generated by addition of 2% galactose. Samples for ChIP on chip were collected and analyzed on ChrVI arrays after 2 hr of galactose incubation. (B) Wild-type (CB499) or temperature-sensitive scc2-4 (CB425) cells expressing Smc6-Flag and containing GAL:HO and the HO cutsite as in (A) were arrested in G2/M when HO was induced by galactose addition to the cultures. Thirty minutes later, the temperature was raised from 23°C to 37°C, and after 1.5 hr, samples for ChIP on chip were collected and analyzed on ChrVI arrays. Annotations as in Figure 4. Molecular Cell 2006 22, 755-767DOI: (10.1016/j.molcel.2006.05.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 Smc6 Malfunction Perturbs Sister Chromatid Separation in the Centromeric Region and Leads to the Accumulation of Branched Chromosome Structures (A) SMC6 (CB67) or smc6-56 (CB308) cells were arrested in G1 by growth in the presence of α factor at 23°C for 2.5 hr. Temperature was then raised to 35°C for 30 min under maintained G1 arrest. Cells were released from G1 at the restrictive temperature, and samples for cell cycle analysis by FACS were collected every 15 min. Fresh α factor was added to the cultures 45 min after release to halt cell cycle progression after mitosis. FACS profiles of selected time points are shown. (B) SMC6 (CB67) or smc6-56 (CB308) cells were arrested in G2/M at 23°C. The temperature was then raised to 35°C for 30 min. Subsequently, cells were released in 35°C medium containing α factor for arrest in G1. Samples for cell cycle analysis and budding index were collected as in (A). FACS profiles from selected time points are shown. (C) Percentage of budded cells analyzed in the same samples as in (A). (D) The decrease in percentage of large budded cells was determined in the same samples used for cell cycle analysis in (B). (E) SMC6 (CB532) or smc6-56 (CB551) cells containing the GFP-Lac-repressor/Lac-operon system for detection of sister chromatid separation 1.8 kb downstream of CENXV were arrested in G1 and the temperature increased from 23°C to 35°C for 30 min. Then, cells were released into the cell cycle at restrictive temperature, and samples for determination of budding index and for analysis of chromatid separation at CENXV were collected every 10 min. At least 200 cells/time point were scored for determination of sister chromatid separation. (F–H) SMC6 (CB67) or smc6-56 (CB308) cells were treated as in (E). Samples for analysis by (F) FACS or (G) PFGE and Southern blot using a ChrXVI probe were collected every 15 min for 2 hr after release from G1 arrest at the restrictive temperature. (H) The Southern blot was quantified by calculating the percentage of the total amount of signal in each sample that remained in the well after PFGE. Molecular Cell 2006 22, 755-767DOI: (10.1016/j.molcel.2006.05.014) Copyright © 2006 Elsevier Inc. Terms and Conditions