1. Gradual Implementation of the Meiotic Recombination Program via Checkpoint Pathways Controlled by Global DSB Levels 
Neeraj Joshi, M. Scott Brown, Douglas K. Bishop, G. Valentin Börner 
Molecular Cell 
Volume 57, Issue 5, Pages (March 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 57, 797-811DOI: (10.1016/j.molcel.2014.12.027)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1
Gradual Establishment of Homolog Bias during Wild-Type Meiosis
(A) Top: map of the HIS4LEU2 locus. The homologous chromosomes Dad and Mom carry XhoI (X) polymorphisms. Probe 4 detects Mom, Dad, DSBs, crossovers (CO-1, CO-2), and JMs, comprising IH-SEI-1 and IH-SEI-2, IS-SEIs, IH-dHJs, and dHJs between two Mom (IS-dHJ(MM)) or two Dad chromatids (IS-dHJ(DD)). Only prominent IH-SEI and IS-SEI species are shown. Bottom: representative sample (left, WT, t = 5 hr) and schematic 2D gel (right). Blue, IH intermediates; red, IS-dHJs; pink, IS-SEIs; asterisks, crosshybridizing species.
(B) Excerpts from the 2D gel analysis of meiotic culture at 33°C from the earliest time point of JM detection (early), and after >70% of CO formation (late). See Figure 1A for peak levels (max). Data in Figures 1A–1F are from meiotic time course (tc) 24. For complete gels, see Figures S1A and S1B. Inverted triangles here and in the supplemental figures indicate early (orange), max (blue), and late (purple).
(C) Quantitation of DSBs, JMs, and COs at HIS4LEU2. Dotted line, DSB peak at 33°C.
(D) Quantitation of JMs as a percentage of the total hybridization signal.
(E) IH-dHJ and IS-dHJ signals as a percentage of respective peak values.
(F) Ratios of IS-dHJ (sum of IS-dHJ(MM) plus IS-dHJ(DD)) to IH-dHJ signals (= IS:IH-dHJ, left y axis). Cumulative levels of dHJs (gray, right y axis) are derived from noncumulative IH-dHJ levels (Supplemental Experimental Procedures). Dashed arrows mark cumulative dHJ levels at the last time point before and the first time point after the minimum IS:IH-ratio is reached.
(G) Average IS:IH-dHJ ratios from multiple cultures. Error bars show SD. The horizontal dashed line indicates the 1:2 ratio expected for no partner preference. Significance was assessed using a two-tailed Student’s t test for correlated samples. ∗∗∗p < 0.001; n.s., not significant, p > 0.05.
(H) Analysis of JMs in ndt80Δ (33°C). For the complete blot, see Figure S1D (right).
(I) Quantitation of dHJs in ndt80Δ. Error bars show SD.
(J) IH-dHJs and IS-dHJs (Figure 1I) as a percentage of maximum values.
(K) Ratios of IS-dHJs to IH-dHJs in ndt80Δ. Error bars show SD. “Entry dHJ” (gray) shows average levels of total dHJs in ndt80Δ as a percentage of maximum levels.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Incomplete Homolog Bias at Low Global DSB Levels during Early Wild-Type Meiosis
(A) Surface-spread nuclei immunodecorated with antibodies against Rad51 and Zip1 (see text for details).
(B) Association of Rad51 with YFP-tagged HIS4LEU2. Shown are representative class A and class B nuclei with the most proximal Rad51 focus at a distance of ≤300 nm (top) or >300 nm from YFP (bottom). Percentages of observed (ob) frequencies and expected frequencies based on a random simulation (ex) are indicated. The insets show magnifications of an ∼400 nm radius around the YFP mark.
(C) Frequency of class A (4–16 Rad51) and class B (≥17 Rad51) nuclei with the closest Rad51 focus ≤300 nm from the YFP mark. Shown are observed (yellow plus gray) and expected (i.e., simulated; gray only) frequencies. N, independent time courses; n, total number of nuclei analyzed. Error bars show SEM.
(D) Nucleus-wide Rad51 focus abundance and recombination at HIS4LEU2. Shown are DSBs at HIS4LEU2 (left y axis), class A through class D nuclei (right inner y axis), and IS:IH-dHJ ratios (right outer y axis, magenta). The average DSB lifespan of t = 49 min (±12 min SD, dotted line) is derived from multiple cultures at 33°C (n = 10, see Experimental Procedures).
(E) Rad51 foci and recombination at HIS4LEU2 in a single meiotic culture. Rad51+ (red) comprises nuclei with ≥4 Rad51 foci (classes A, B, and C). DSBs, dHJs, and COs at HIS4LEU2 are indicated as a percentage of maximum levels. Nuclei are n = 71 (1.66 hr), 74 (2 hr), 39 (2.33 hr), 93 (2.66 hr), 44 (3 hr), and 71 (4 hr).
(F) Excerpts from 2D gel Southern blot analysis. Asterisk, t = 2.66 hr (shown for better visualization). For the complete blot, see Figure S1D (left).
(G) DSBs and COs at HIS4LEU2 in meiotic culture also analyzed for Rad51 and Zip1. For quantitation, see Figure S2A (tc 22).
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5. Figure 3 Control of Homolog Bias by Global DSB Levels
(A) Quantitation of DSBs (left) and COs (middle) at HIS4LEU2 in parallel WT and spo11 hypomorphic cultures. For gels, see Figure S2C. Right: DSBs as a percentage of maximum levels.
(B) Excerpts from 2D gel Southern blot analysis at HIS4LEU2. For blots and reproducibility, see Figures S2A and S3. Asterisk, t = 3.3 hr (shown for clarity, although IS:IH ratios are similar at t = 2.8 hr). Higher levels at early times of the (left) IS-dHJ (MM) versus the (right) IS-dHJ (DD) signal are likely due to earlier onset of DSB formation on the Mom allele (see Figure S2C).
(C) IH-dHJs and IS-dHJs in three spo11 variants.
(D) Ratios of IS-dHJs to IH-dHJs in three spo11 variants from early to late. Inverted triangles refer to the excerpts in (B). See also Figures S2D and S3E.
(E) Average IS:IH ratios at JM appearance (early), peak levels (max), and disappearance (late) at 33°C. Error bars show SD. A two-tailed Student’s t test for correlated samples was used to assess the significance of differences. ∗p < 0.05; n.s., p > 0.05.
(F) Quantitative analysis of dHJs in spo11-HA/yf ndt80Δ. Top: IH-dHJs and IS-dHJs as a percentage of total DNA. Error bars show SD. Samples after t = 7 hr were excluded from analysis because of continued, ndt80Δ-specific DSB formation (Allers and Lichten, 2001). Bottom: average ratios of IS-dHJs to IH-dHJs in spo11-HA/yf ndt80Δ and SPO11 ndt80Δ (from Figure 1K). Kinetics of dHJs in spo11-HA/yf ndt80Δ are shown in gray as a percentage of levels at t = 7 hr. Error bars show SD.
(G) Analysis of JMs in spo11-HA/yf ndt80Δ at time of first detection (t = 4 hr) and at maximum informative levels (t = 7 hr). Compare this with Figure 1H. For the complete blot, see Figure S1D (right).
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6. Figure 4
PCH2 Mediates Homolog Bias of Early-Occurring, Low-Abundance DSBs
(A) Analysis of JMs in PCH2 and pch2Δ in (i) SPO11, (ii) spo11-HA, and (iii) spo11-HA/yf backgrounds. Asterisk, t = 5 hr (shown for better visualization of IS-dHJs in spo11-HA PCH2). For complete blots, see Figure S3.
(B) Role of PCH2 in IH recombination during early SPO11 meiosis. Left: Ratios of IS-dHJs to IH-dHJs (IS:IH-dHJ) in PCH2 and pch2Δ from earliest appearance to late time points. Right: Average IS:IH-dHJ ratios in multiple cultures. Error bars show SD.
(C) IS:IH-dHJ ratios and quantitation of JMs in the WT (blue) and pch2Δ (red) at normal and reduced DSB levels (SPO11, left; spo11-HA, center; spo11-HA/yf, right). See also Figures S4A and S4C).
(D) Average IS:IH-dHJ ratios from multiple WT and pch2Δ cultures at normal and reduced DSB levels at time of appearance (early), peak levels (max), and disappearance (late). Error bars show SD.
(E) Recombination at HIS4LEU2 in PCH2 or pch2Δ in SPO11, spo11-HA, and spo11-HA/yf backgrounds. For blots, see Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
PCH2 Mediates Homolog Bias for Early-Replicating Genome Segments
(A) ARS: in the presence of the three active replication origins on the left arm of chromosome III (black circles, ARS ), HIS4LEU2 is frequently replicated from a proximal origin with ensuing early DSB formation (yellow lightning bolts). arsΔ-LIII: Deletion of ARS305, ARS306, and ARS307 (circled Xs) results in passive replication of LIII from the right chromosome arm, with DSBs at HIS4LEU2 occurring with a delay, at higher global DSB levels (Figures S5A and S5B).
(B) Analysis of JMs at HIS4LEU2 in the presence (ARS, left) or absence of replication origins (arsΔ-LIII, right) in PCH2 or pch2Δ. For blots, see also Figure S5C.
(C) Analysis of JMs at YCR047c in cultures also analyzed at HIS4LEU2 (Figure 5B). Restriction digestion was performed with HindIII. For blots, see Figure S5D.
(D) Delayed replication reduces dependence on PCH2 of early homolog bias. Left: IS:IH-dHJ ratios in PCH2, pch2Δ, and pch2Δ arsΔ-LIII. Right: average IS:IH-dHJ ratios in multiple cultures. Error bars show SD.
(E) Rapid homolog bias establishment during WT meiosis because of delayed replication. Left: Ratios of IS-dHJs to IH-dHJs in WT in ARS and arsΔ-LIII. Right: average IS:IH-dHJ ratios in multiple ARS or arsΔ-LIII cultures. Error bars show SD.
(F) Delayed replication results in increased COs in pch2Δ and PCH2 in individual cultures. Measurements right of the dashed separator line indicate average maximum COs (± SD) from multiple cultures.
(G) Comparison of COs at HIS4LEU2 in pch2Δ and PCH2 cultures with (ARS) or without (arsΔ-LIII) functional replication origins along LIII. Samples are from tc 27 (pch2Δ) and tc 31 (PCH2). Average CO levels (± SD) from multiple cultures as a percentage of the total hybridization signal are shown below the blots. P values indicate significances from two-tailed Student’s t tests for correlated samples.
(H) Quantitation of dHJs at HIS4LEU2 (top) and YCR047c (bottom) in pch2Δ (left) or PCH2 (right) strains with (ARS) or without (arsΔ) functional replication origins along LIII.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
Distinct and Overlapping Roles in Interhomolog Recombination of PCH2, MEC1, and TEL1
(A) Analysis of JMs in mec1-kd and pch2Δ mec1-kd. For complete blots, see Figure S6C.
(B) Analysis of JMs in tel1Δ and pch2Δ tel1Δ. For complete blots, see Figure S6D.
(C) Analysis of JMs in TEL1 and tel1Δ in a spo11-HA/yf background. For complete blots, see Figure S6H.
(D) Overlapping functions of PCH2 and MEC1 in IH recombination. Shown are average IS:IH-dHJ ratios from multiple mec1-kd and pch2Δ mec1-kd cultures. Error bars show SD.
(E) Quantitative analysis of DSBs and COs in PCH2 (blue) and pch2Δ (magenta) in a mec1-kd sml1Δ background. For the blot, see Figure S6A.
(F) Overlapping functions of PCH2 and MEC1 in IH recombination. Top: ratios of IS-dHJs to IH-dHJs (IS:IH) in PCH2 mec1-kd (blue) and pch2Δ mec1-kd (magenta). Bottom: Quantitation of IH-dHJs and IS-dHJs.
(G) Effects of tel1Δ on IH recombination in SPO11. Top: ratios of IS-dHJs to IH-dHJs (IS:IH-dHJ) in parallel meiotic cultures carrying PCH2 tel1Δ (blue), pch2Δ tel1Δ (red), pch2Δ TEL1 (black), and PCH2 TEL1 (gray). Bottom: levels of IH-dHJs and IS-dHJs in cultures shown at the top. pch2Δ TEL1 is omitted for clarity.
(H) Role of TEL1 in homolog bias at reduced DSB levels. Top: ratios of IS-dHJs to IH-dHJs (IS:IH) in TEL1 and tel1Δ in a spo11-HA/yf background. Bottom: IH-dHJs and IS-dHJs from cultures shown at the top.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
TEL1 Mediates Resection of Early-Occurring, Low-Abundance DSBs
(A) DSB resection at HIS4LEU2 in tel1Δ, pch2Δ, pch2Δ tel1Δ, and the WT. Shown are excerpts from the 1D gel Southern blot analysis of genomic DNA digested with PvuII, generating a single fragment. HIS4LEU2 (P), parental band; arrows, hyporesected and normally resected DSBs; arrowhead, crosshybridizing band with invariable migration position. For complete blots, see Figure S7A.
(B) Lane intensity profiles of tel1Δ (red) and WT (black) at t = 2 hr, used for quantitating resection differences (Figures 7D and 7E).
(C) DSB resection in tel1Δ and TEL1 at reduced DSB levels. For details see legend Figure 7A. For complete blots see Figure S7B.
(D) Quantitation of DSB resection in tel1Δ, pch2Δ tel1Δ, and TEL1 at full DSB levels (SPO11). Left: ratios (mutant/WT) of distance measurements between the intensity peaks of the parental and DSB bands. Dashed line, expected result for identical resection in the mutant and WT. Right: DSB kinetics for the cultures on the left. The WT is shown as a dashed line.
(E) Quantitation of DSB resection in tel1Δ and TEL1 in three spo11 variants. Left: DSB resection in spo11-HA/yf (see Figure 7C). Right: analysis of DSB resection in WT and tel1Δ cultures carrying SPO11, spo11-HA, and spo11-HA/yf (see Figures S7D–S7F).
(F) Model for sequential roles of Tel1ATM/ Pch2 and Mec1ATR in recombination control at lower and higher DSB levels during WT meiosis. Homolog bias and sister bias are signified by a blue or a pink arrow, respectively. Darker arrows indicate a stronger bias. See text for details.
Molecular Cell  , DOI: ( /j.molcel )
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