1. Chromosome-wide Rad51 Spreading and SUMO-H2A
Chromosome-wide Rad51 Spreading and SUMO-H2A.Z-Dependent Chromosome Fixation in Response to a Persistent DNA Double-Strand Break 
Marian Kalocsay, Natalie Jasmin Hiller, Stefan Jentsch 
Molecular Cell 
Volume 33, Issue 3, Pages (February 2009)
DOI: /j.molcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1 Recruitment of H2A.Z to a DNA DSB
(A) Schematic representation of chromosome III. A single DSB (arrow) is created at the MAT locus by expression of HO endonuclease. Regions of homology are shown as boxes. HML and HMR homologous sequences are deleted in strain JKM179 (Δ). Positions of primer pair PMAT spanning the break site used for assaying DSB formation and primer pair P+0.2 used for H2A.Z-directed ChIP are indicated. A filled circle denotes the relative position of the centromere.
(B) DSB formation determined by quantitative PCR (primer pair PMAT) over time. Continuous expression of HO prevents repair by NHEJ and sister chromatid recombination.
(C) H2A.Z is recruited early but transiently to the DSB. Htz13HA-directed ChIP at 0.2 kb (primer pair P+0.2) from the HO cut site. Shown are IP/input signals normalized to 1 for the signal before induction.
(D) H2A.Z is incorporated into the right flank of the DSB. Htz13HA-directed ChIPs at indicated time points (as in [C]) were amplified and hybridized to high-density whole genome tiling arrays (ChIP-on-chip). H2A.Z enrichments (IP/input) were normalized to the 0 hr data set to obtain the fold enrichment in H2A.Z-directed ChIP signals after DSB induction (for details, see Figure S2). The position of the DSB (HO cut site) is indicated by a red arrow. Data in (B) and (C) are represented as mean ± SEM.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

3. Figure 2 H2A.Z Mutant Phenotypes
(A) Defect of mutants deficient in H2A.Z in DNA resection as measured by the loss of DNA at the region flanking the DSB (Chen et al., 2008). Shown is the loss of input DNA levels at MAT, 0.2 kb from the DSB, in WT (triangles) and Δhtz1 (squares). The 0 hr time point signal was set to 100%.
(B) Defect of mutants deficient in H2A.Z as measured by Southern blot analysis of 5′ strand resection (Zhu et al., 2008). Genomic DNA prepared from samples taken at the indicated time points was digested with EcoRI and run on an alkaline gel followed by gel blotting and hybridization with strand-specific ssRNA probes as indicated. The SNT1 probe binds 10 kb to the left of the break; the loading control is a probe against the unaffected TRA1 locus on chromosome VIII. 5′-strand resection progressively eliminates the EcoRI cut sites around the break site; therefore, the 5.3 kb SNT1 fragment disappears in WT after 6–8 hr.
(C) Rfa16HA-directed ChIP at the indicted distances from the DSB and at the indicated time points after HO induction in WT (left panel) and Δhtz1 (right panel). The heat bar indicates relative enrichments (IP/input).
(D) Defective checkpoint activation monitored by Rad53 phosphorylation in Δhtz1 and Δswr1 compared to WT.
(E) Defective checkpoint activation monitored by γH2AX (phospho-H2A)-directed ChIP at 9.5 kb from the DSB in WT and Δhtz1. Shown are IP/input signals normalized to 1 for the signal before induction. Data in (A) and (E) are represented as mean ± SEM.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

4. Figure 3 Rad51 Spreading along the DSB-Harboring Chromosome
(A) Rad51-directed ChIP-on-chip analysis (genome-wide), 2 hr after HO-induced DSB formation on chromosome III. Shown are approximately 380,000 IP/input signals on a linear scale aligned to their relative position in kilobases on each yeast chromosome.
(B) As above (only chromosome III is shown), but ChIPs were done at the indicated time points after DSB induction. Chromosome III is shown enlarged with the position of the persisting DSB at kb (red line). The asterisks label homology present on the tiling array, which is, however, deleted in the strain used. Single spikes correspond to single oligonucleotide data and are hybridization artifacts. Gaps in the Rad51 profile correspond to, e.g., Ty1 transposons (e.g., at 150 and 170 kb), which, because of their repetitiveness, cannot be analyzed. However, note that Rad51 spreading slows down past these elements. In both panels, filled circles denote the position of centromeres.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

5. Figure 4
Recruitment of a Persistent DSB to the Nuclear Periphery and Dependency on H2A.Z, Rad51, and the DNA-Damage Checkpoint
(A) In vivo visualization of DSB recruitment to the nuclear periphery. Expression of a GFP-LacI fusion marks a 240× lacO array (green) integrated proximal to MAT. The nuclear envelope is visualized by Nic96mars (red). Colocalization was scored in cells 0 or 5 hr after HO endonuclease induction (mean and SEM of three independent experiments, n > 345). The insets show examples for nucleoplasm and nuclear periphery localization of the GFP signal.
(B) Mps39myc-directed ChIP at the MAT locus (0.2 kb from the DSB) after HO induction. Shown are IP/input signals normalized to 1 for the signal before induction.
(C) Mps39myc-directed ChIP-on-chip of chromosome III, showing that Mps39myc is enriched at the DSB, 5 hr after induction. The asterisks label homology present on the tiling array, which is, however, deleted in the strain used. Mps39myc enrichments (IP/input) were normalized to the 30 min data set.
(D) Similar to (B), but here Mps39myc-directed ChIP data of WT and the indicated mutants were compared. The right panel shows Rad53 phosphorylation as an indicator of checkpoint activation.
(E) Similar to above, but here cells were arrested with α factor in G1. Quantification of at least three independent experiments. Data are represented as mean ± SEM.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

6. Figure 5
H2A.Z SUMOylation Is Required for Break Recruitment to the Nuclear Periphery
(A) Yeast H2A.Z is modified by SUMO, giving rise to species carrying one (H2A.Z1xSUMO) or two (H2A.Z2xSUMO) SUMO moieties. Proteins modified by His-tagged SUMO were isolated by denaturing NiNTA pull-down, and 3HA-tagged H2A.Z was identified by blotting with HA-epitope-specific antibodies. Dpm1 was used as input control. Controls C1 and C2 do not express tagged H2A.Z or SUMO, respectively. Compared were WT, single, and double lysine mutant H2A.Z proteins (K126R, K133R, and K126,133R) and a mutant variant that lacks all lysines in the C-terminal tail (C-term. K0).
(B) SUMOylation-defective H2A.Z variant (htz1K126R) is incorporated at the DSB similar to the WT H2A.Z protein shown in Figure 1D.
(C) Mps39myc-directed ChIP as in Figure 4D, but in SUMOylation-defective htz1 mutants.
(D) Quantification of live-cell microscopy data of DSB relocalization to the nuclear periphery (similar setup as that in Figure 4A, n > 550). Shown are means of three independent experiments ± SEM.
(E) SUMOylation-defective htz1 mutants do not exhibit defects in checkpoint activation after HO induction and DSB formation as monitored by Rad53 phosphorylation.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

7. Figure 6
Choreography of Chromosomal Events after Induction of a Persistent DSB
Summary of the data from Figures 1D, 3B, and 4C. The position of the DSB is indicated by a red arrow. To obtain the fold enrichment, ChIP data (IP/input) were normalized to the respective 0 hr data sets, and for Mps39myc (nuclear periphery) to the 30 min data set. Z axis is on a log10 scale.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions