Presentation is loading. Please wait.

Presentation is loading. Please wait.

The Smc5/6 Complex Is an ATP-Dependent Intermolecular DNA Linker

Similar presentations


Presentation on theme: "The Smc5/6 Complex Is an ATP-Dependent Intermolecular DNA Linker"— Presentation transcript:

1 The Smc5/6 Complex Is an ATP-Dependent Intermolecular DNA Linker
Takaharu Kanno, Davide G. Berta, Camilla Sjögren  Cell Reports  Volume 12, Issue 9, Pages (September 2015) DOI: /j.celrep Copyright © 2015 The Authors Terms and Conditions

2 Cell Reports 2015 12, 1471-1482DOI: (10.1016/j.celrep.2015.07.048)
Copyright © 2015 The Authors Terms and Conditions

3 Figure 1 The Purified Smc5/6 Fraction Contains Topoisomerase-like Activities (A) Oriole-stained polyacrylamide gel of purified Smc5/6 from wild-type cells. Bands indicated in red (1–6) were subjected to mass spectrometry (see Table S1). (B–F) Southern blot analysis of samples treated as follows. (B) Indicated molar ratios of Smc5/6 were incubated with 30 pM SC(−) plasmid for 90 min. (C) Schematic diagram for 2D gel migration of topoisomers (left). Analysis of 2D gel of SC(−) (top) and relaxed (bottom) plasmids after mock treatment or incubation with Smc5/6 (protein to DNA ratio 200:1) as indicated. CHL, chloroquine. (D) Smc5/6 was incubated with SC(−) plasmid for indicated times at a protein/DNA molar ratio of 200. (E) Smc5/6 was incubated with indicated forms of plasmids (protein to DNA ratio 200:1) in the absence or presence of 100 μM ATP or ATPγS (γS). Left: pRS316-derived plasmid. Right: pBR322-derived plasmid. (F) 2D gel analysis performed as in (C) with pBR322-derived SC(−) (left) and SC(+) (right) plasmids. In (B), (D), and (E), the migration pattern for catenanes, relaxed, linear, and SC(−) plasmids are indicated. See also Figure S1. Cell Reports  , DOI: ( /j.celrep ) Copyright © 2015 The Authors Terms and Conditions

4 Figure 2 The Smc5/6 Complex Co-purifies with Top1 and Top2
(A) Oriole-stained polyacrylamide gels of purified Smc5/6 from top1Δ, top2-4 ts or top3Δsgs1Δ cells as indicated. (B–E) Southern blot analysis of samples treated as follows. (B) Smc5/6 from top3Δsgs1Δ cells were incubated with 30 pM of SC(−) or relaxed pRS316-derived plasmids at indicated ratios. (C and D) Smc5/6 from top1Δ or top2-4 ts cells was incubated with SC(−) or relaxed plasmids as in Figure 1E. For samples containing Smc5/6 from the top2-4 ts strain (D), the incubation was performed at 35°C, which is the restrictive temperature for top2-4 in vivo. (E) An aliquot of 0.5 μl from a mock purification using cells lacking TAP-tagged protein was incubated for 90 min with 30 pM SC(−) plasmid in the presence of indicated amounts of Smc5/6 from top1Δ cells. (F) Smc6 was isolated using FLAG immunoprecipitation from Top2-HA-expressing cells (see the Supplemental Experimental Procedures for details), and samples were analyzed by western blot using an α-HA antibody (top) or an α-FLAG antibody (middle). The bottom panel displays the protein gel stained with Stain-Free system (Bio-Rad), which is shown as a loading control. WCE, whole-cell extract; IP, immunoprecipitated fraction. Cell Reports  , DOI: ( /j.celrep ) Copyright © 2015 The Authors Terms and Conditions

5 Figure 3 Stimulation of Top2-Dependent Catenation by Smc5/6 Requires the Complex’s ATPase Activity (A) Southern blot analysis with α-TAP antibody of relaxed, Smc5/6 treated pRS316-derived plasmid. Before incubation with the plasmid, Smc5/6 was pre-incubated with increasing amounts of an α-TAP antibody (0.5, 2.5, or 5.0 ng/μl) or a control α-myc antibody (5.0 ng/μl). (B) Indicated amounts of human TOP2α were pre-incubated for 15 min at 30°C with or without 6 nM Smc5/6 from top2-4 ts cells before incubation with 30 pM relaxed plasmid for 90 min at 35°C. A reaction with Smc5/6 from wild-type strain is also shown (wt). Samples were then treated with proteinase K and heat-denatured before Southern blot analysis. (C) Left: experimental setup. Right: Southern blot analysis of plasmids treated as indicated. First, Smc5/6 from the top2-4 ts strain was incubated without addition of nucleotide or with 1 mM of ATP (P) or ATPγS (S) for 60 min at 35°C. Subsequently, 50 mU human TOP2α and 1 mM of ATP (P) and/or ATPγS (S) were added, and the reaction was stopped using proteinase K treatment and heat-denaturation after 5 min incubation at 35°C. The nature of fussy signals on the lower part of the gel in (B) and (C) is unknown but likely due to the heat denaturation of DNA. See Figure 1E for comparison. See also Figure S2. Cell Reports  , DOI: ( /j.celrep ) Copyright © 2015 The Authors Terms and Conditions

6 Figure 4 Smc5/6 Binds Plasmid DNA under Low-Salt Conditions
(A–C) Increasing amounts of Smc5/6 from top1Δ cells (protein/DNA molar ratios 40, 80, and 200) were incubated with SC(−), linear, or nicked plasmids for 15 min at 30°C, and their binding to DNA was investigated using a filter binding assay. Each experiment was repeated three times, and mean values and SDs are indicated. (A) DNA binding to pRS316-derived substrates as indicated. (B) DNA binding to pBR322-derived substrates as indicated. (C) Comparison of DNA binding for pRS316 SC(−), pBR322 SC(−), and pBR322 SC(+) at a protein/DNA molar ratio of 200. No significant difference was detected using t test analysis. Cell Reports  , DOI: ( /j.celrep ) Copyright © 2015 The Authors Terms and Conditions

7 Figure 5 The Smc5/6 Complex Binds DNA through Direct Interactions and ATP Hydrolysis-Dependent Topological Entrapment (A) Each substrate (SC(−), linear, and nicked) was incubated with Smc5/6 from top1Δ cells for 15 min at 30°C and applied to glass fiber filters. Flow through (FT), wash and elution fractions using 3 × 50 μl reaction buffer (W1–W3), 3 × 50 μl reaction buffer supplemented with 1 M NaCl (H1-3), and 3 × 50 μl 0.5% SDS buffer (S1–S3) were collected and subjected to Southern blot analysis. Results from analysis of nicked plasmid using the same experimental setup but with buffers without ATP are shown in the lowermost panel. (B) Left: schematic figure of experimental setup. Right: Southern blot analysis of nicked plasmid after pre-incubation with Smc5/6 for 15 min at 30°C, and subsequent linearization using HindIII before analysis by filter binding assay as in (A). Fractions were combined before loading to the agarose gel as follows: W, FT and W1–W3; H, H1–H3; and S, S1–S3. (C) Smc5/6 from a top1Δ strain (4 nM) was incubated with 30 pM nicked plasmid in the absence or presence of ATP or ATPγS, and subjected to filter binding assay and Southern blot analysis as in (A). Left: representative Southern blot. Right: quantification. Mean values of the ratios of SDS elution fractions from three independent experiments are shown. SDs and p values (t test, ∗p < 0.05) are indicated. Cell Reports  , DOI: ( /j.celrep ) Copyright © 2015 The Authors Terms and Conditions

8 Figure 6 An Smc5/6K115E Complex Displays Reduced ATPase Activity, DNA Binding, Chromosome Association, and Stimulation of TOP2α-Dependent Catenation (A) Oriole-stained polyacrylamide gel of purified Smc5/6K115E. (B and C) ATPase activity of 6 nM Smc5/6 (B) and Smc5/6K115E (C) from otherwise wild-type cells using indicated molar ratios of pRS316. The experiments were repeated three times, and mean values and SDs are indicated. (D) DNA binding of Smc5/6K115E from top1Δ strain measured as in Figure 4A. (E) Chromosomal association of SMC6- and SMC6K115E-FLAG, cloned into the TRP1 locus under the control of their endogenous promoters, at selected chromosomal positions determined by ChIP-qPCR. Cells were arrested in G1 or G2/M (G2) phase before sample preparation. Chromosome number and distance in kb from the left telomere of each analyzed position is indicated. y axis displays the amount of DNA in ChIP fraction in relation to input. Mean values from three independent experiments are shown. SDs and p values (t test, ∗p < 0.05) are indicated. (F) Smc5/6 (W) and Smc5/6K115E (K) from top2-4 ts cells were pre-incubated with 30 pM relaxed plasmid for 60 min at 35°C in reaction buffer containing 1 mM ATP. Then indicated amounts of human TOP2α were added, and incubated for 5 min before the reaction was stopped using proteinase K treatment and heat-denaturation. As in Figures 3B and 3C, the nature of the fussy signal in the lower part of the gel is unknown. Cell Reports  , DOI: ( /j.celrep ) Copyright © 2015 The Authors Terms and Conditions

9 Figure 7 Overexpression of the smc6K115E ATPase Motif Mutant Inhibits Cellular Growth and Cell-Cycle Progression and Activates the DNA Damage Checkpoint (A–D) Cells harboring GAL1-10 (control), GAL1-10:SMC6 or GAL1-10:SMC6K115E in the TRP1 locus were analyzed as follows. (A) Serial dilutions (10× dilutions; 100–10−5) of the indicated cell types grown on YEP plates containing glucose or galactose for 2 days at 30°C. (B) Growth curve. Logarithmically growing cells were diluted in YEP medium with or without galactose (+gal/−gal), and cell density was measured at indicated time points. (C) Fluorescence-activated cell sorting (FACS) profiles. Cells were arrested in G1 phase and released into YEP medium with or without galactose. Samples were collected at indicated time points and processed for FACS analysis. (D) Western blot of Rad53. Logarithmically growing cells were diluted in galactose containing YEP medium with 200 mM hydroxyurea (HU) (left) or without HU (right). Samples were collected at indicated time points, and Rad53 was detected by western blot using an α-Rad53 antibody. The upshifted band represents the activated, phosphorylated form of Rad53 (Rad53∗). The HU-treated samples show that all strains are able to activate Rad53 upon HU-induced replication block. Cell Reports  , DOI: ( /j.celrep ) Copyright © 2015 The Authors Terms and Conditions


Download ppt "The Smc5/6 Complex Is an ATP-Dependent Intermolecular DNA Linker"

Similar presentations


Ads by Google