1. Volume 47, Issue 5, Pages 722-733 (September 2012)
A Central Coupler for Recombination Initiation Linking Chromosome Architecture to S Phase Checkpoint 
Tomoichiro Miyoshi, Masaru Ito, Kazuto Kugou, Shintaro Yamada, Masaki Furuichi, Arisa Oda, Takatomi Yamada, Kouji Hirota, Hisao Masai, Kunihiro Ohta 
Molecular Cell 
Volume 47, Issue 5, Pages (September 2012)
DOI: /j.molcel
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2. Molecular Cell 2012 47, 722-733DOI: (10.1016/j.molcel.2012.06.023)
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 1
Two Distinct Expression Patterns of Rec12 Partners during Meiosis
(A) Protein expression patterns of Rec12 partners during meiosis. Cells were meiotically synchronized and harvested at the indicated time points. Whole-cell extracts were prepared and analyzed by immunoblotting.
(B) DNA content was analyzed by flow cytometry.
(C) Relative protein amounts normalized to control signals (alpha-tubulin) were calculated using ImageQuant software.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Mde2 Is a Cofactor Stabilizing the SFT Subcomplex and Interacts with the DSBC Subcomplex
(A) Protein-protein interactions were analyzed by a yeast two-hybrid assay.
(B) Mde2 interacts with Rec14. Cells were meiotically synchronized and treated with a crosslinker (0.9% formaldehyde) at 4 hr after meiotic induction (left panel). GST-Mde2 (REP1) and Rec14-FLAG (REP2) were coexpressed in wild-type cells (right panel). Then, the extracts were subjected to coimmunoprecipitation after the MNase treatment or GST pull-down assay. Asterisk indicates nonspecific bands. Arrows indicate the positions of Mde2-FLAG, Rec14-HA, and Rec14-FLAG, respectively.
(C) Co-occupancy of Mde2 and Rec14 at the DSB hotspot. Meiotically synchronized cells were fixed at 4 hr after meiotic induction and subjected to sequential ChIP assay (re-ChIP) according to the method described elsewhere (Ishizaka et al., 2012). The primary immunoprecipitated chromatin with anti-FLAG antibody was followed by the secondary ChIP with anti-HA antibody. IP efficiencies were measured by quantitative PCR using primer sets for sib1 (cold) or mbs1 (DSB hotspot).
(D) Mde2 is not essential for the assembly of the DSBC subcomplex. WCEs were prepared at 4 hr after meiotic induction in wild-type or mde2Δ. IPs were obtained from WCEs using the indicated antibodies and analyzed by immunoblotting.
(E) Rec15 interacts with Mde2. Cells were meiotically synchronized. Immunoprecipitation and immunoblotting were performed as in (D). Asterisk indicates nonspecific bands. An arrow indicates the positions of Mde2-HA.
(F) Co-occupancy of Mde2 and Rec15 at the DSB hotspot. Re-ChIP was performed as in (C).
(G) Mde2 is essential for the assembly of the SFT subcomplex. Immunoprecipitation and immunoblotting were performed as in (D).
(H) Summary of the network for protein-protein interactions among DSB proteins. The yeast two- or three-hybrid assay, fluorescence microscopy, and coimmunoprecipitation analyses support interactions indicated by dashed lines. See also Figure S1 and Table S1.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
Rec15 Binds to LinE sites or DSB Hotspots in a Rec10- or Rec24 (Mde2)-dependent Manner, Respectively
(A) Yeast two-hybrid assay testing all the pairwise interactions of DSB proteins with four LinE components.
(B) ChIP-chip analyses of Rec15 and Rec10. Cells were fixed at 4 hr after meiotic induction and subjected to ChIP-chip analysis. The horizontal axis represents the physical position on the chromosome. The vertical axis represents the signal intensity of Rec15, Rec10, or Rec12 on a log2 scale relative to input DNA signals. Green (Rec10), blue (Rec15), and red (Rec12) vertical bars show the signal ratio of loci significantly enriched in the IP fraction. The horizontal boxes represent the positions of open reading frames. The ChIP-chip data of Rec12 is as previously reported (Hyppa et al., 2008).
(C) Smoothed binding signal of Rec10 around DSB hotspots. All signal log2 ratios (IP/Input) of Rec10 located 10 kb from every DSB peaks were extracted and aligned at DSB peak positions. Smoothing was performed with the “smooth.spline” function in R (
(D) Smoothed binding signal of Rec15 around DSB hotspots (left panels) and LinE sites (right panels). Same as in (C), but no Rec15 signals overlapping with the DSB hotspots were extracted in the right panels.
(E) Stacked bar graphs representing the number of binding loci for Rec15-FLAG associated with Rec10-FLAG only (blue), Rec12-FLAG only (red), and both proteins (yellow). In the column labeled “Rec10 with Rec12,” sites for Rec10, Rec12, or both are indicated in green, red, and yellow, respectively.
(F) MA-plot of the ChIP-chip data on Rec15-FLAG in rec24Δ versus Rec15-FLAG in wild-type. The intensity ratio of Rec15-FLAG in rec24Δ to Rec15-FLAG in wild-type is plotted against its average intensity. The probes located in DSB hotspots (enrichment of Rec12 ≥ 2, in log2; see Supplemental Experimental Procedures) are indicated with red dots. The IP/Input ratio was compared every 500 bp across the genome. The enrichment of Rec12 for each probe represents the average of Rec12 binding signals over 500 bp. y axis (M); Rec15-FLAG in rec24Δ/wild-type intensity ratio (log2), x axis (A); Rec15-FLAG in rec24Δ and wild-type average intensity (log2). See also Figure S2 and Table S2.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Mde2 and Components of the SFT Subcomplex Mediate Binding of Rec10 and Rec15 to DSB Sites
(A) Requirements of the Rec12 partners and axis proteins for Rec15 binding to the mbs1 DSB hotspot and LinE sites. Cells were crosslinked at 4 hr after meiotic induction and subjected to ChIP analysis. IP efficiencies were measured by quantitative PCR using primer sets for sib1 (cold), mbs1 (DSB hotspot), or SPBC3H7.03 loci (LinE/axis site).
(B) Schematic presentation of the locations of the cold region (sib1), DSB hotspots (mbs1/sat1), and LinE/axis loci (SPBC3H7.03/bir1) tested by quantitative PCR.
(C) Rec24 binding to the DSB hotspot and LinE site depends on Rec15. ChIP assay was performed as in (A). Bars represent mean values with SDs.
(D) Requirements of Rec12 partners and Rec8 for the Rec10 binding to DSB hotspots and LinE sites. ChIP assay was conducted as in (A) using primer sets for sib1 (cold), mbs1, and sat1 (DSB hotspots), or SPBC3H7.03 and bir1 loci (LinE/axis sites). See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
Mde2 Is Concertedly Recruited to DSB Sites with the SFT Subcomplex and Is Induced after the Completion of Meiotic DNA Replication
(A) The Mde2 expression is inhibited by a DNA replication inhibitor, HU. G1-arrested cells were synchronously induced into meiosis in the presence or absence of 20 mM HU (hydroxyurea) and harvested at the indicated time points. Whole-cell extracts were prepared and analyzed by immunoblotting (left panel). DNA content of the cells was analyzed by flow cytometry (right panel).
(B) ChIP-chip analysis of Mde2. The ChIP-chip experiment was performed as in Figure 3. Blue (Rec15), gray (Mde2), and red (Rec12) vertical bars show the signal ratio of loci significantly enriched in the IP fraction.
(C) Stacked bar graphs representing the number of binding loci for Mde2 associated with Rec10-FLAG only (gray), Rec12-FLAG only (red), and both proteins (yellow).
(D) Smoothed binding signal of Mde2 around DSB hotspots (left panels) and LinE sites (right panels).
(E) Rec12 is recruited to the DSB hotspot in an Mde2-dependent manner. Cells were crosslinked at 4 hr after meiotic induction and subjected to ChIP analysis. IP efficiencies were measured by quantitative PCR using primer sets for sib1 (cold), mbs1 (DSB hotspot), or SPBC3H7.03 loci (LinE/axis site). See also Table S2.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6 Model Mechanism for the Initiation of DSB Formation
The proposed model for the control of fission yeast DSB formation. See the detailed description in the Discussion.
Molecular Cell  , DOI: ( /j.molcel )
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