Figure 1.
Rec114 is a DSB dependent Tel1/Mec1 target.
A. Schematic representation of Rec114 with the locations of eight [S/T]Q motifs. S: serine, T: threonine, SCD: [S/T]Q Cluster Domain. Below: Slower migrating Rec114 species revealed in Western blot analysis using polyclonal α-Rec114 antibodies. B–D. Samples from indicated genotypes were collected at the specified time points and subjected to a Western blot analysis using α-Myc or α-Hop1 antibodies. E. Samples from REC114 and rec114-8A cultures were collected at 3, 5, and 7 hours after induction of meiosis, and subjected to immunoprecipitation using α-Rec114 antibodies. The resulting precipitates were separated in SDS gels and immunoblotted using three phosphos-specific antibodies (α-pThr175, α-pSer187, α-pSer265), or α-Rec114 antibodies. F. In vitro kinase assay using immunoprecipitated Mec1-myc18 and purified GST-Rec114 and GST-Rec1148A in the presence of “cold” ATP. Samples were separated in SDS gels and immunoblotted using a cocktail of α-pThr175, α-pSer187, and α-Ser265 antibodies or α-Rec114 antibodies. G. Samples from indicated genotypes were collected 5 hours after induction of synchronous meiosis and subjected to Western blot analysis using α-pThr175 or α-Rec114 antibodies.
Table 1.
Spore viability of the different rec114 alleles in various genetic backgrounds.
Figure 2.
Effect of rec114-8A and rec114-8D on levels of COs and DSBs.
A. Physical map of HIS4-LEU2 locus showing relevant XhoI restriction sites (X) and the probe used for Southern analysis [36]. Parental homologs “Mom” and “Dad” and the two CO-products are distinguished via restriction polymorphism (circled X). Sizes and identities of species analyzed in (B) are as indicated. “COs”: interhomolog crossover products. B. Southern blot analysis of COs in REC114, rec114-8A, and rec114-8D strains. The analysis was performed as described in panel A and Materials and Methods. C. Quantification of COs in the gel shown in panel B. D. Mapping of meiotic DSBs in ChrIII by PFGE followed by indirect labeling of one chromosome end using YCL064C/CHA1. FL: full-length intact chromosomes. DSBs: linear chromosome fragments extending from the labeled end to the site of a break. E. PFGE of whole chromosomes probed with the YCL064C/CHA1 probe from REC114, rec114-8D, and rec114-8A strains in a com1Δ/sae2Δ background; the region of the gel used for DSB quantification is indicated by brackets on the right of the gel. Quantitative analysis of the PFGE/Southern gel is presented below. F. Southern blot analysis of the region around the YCR047C YCR048W DSB-hotspot. Samples were digested with AseI restrictive enzyme and probed with YCR048W to assess DSB levels in a REC114, rec114-8A, or tel1Δ strain in a rad50S background. Quantitative analysis was performed based on the signal associated with the DSB-hotspot located within the YCR047C promoter (*). G. PFGE of whole chromosomes probed with the YHL039W probe from REC114, rec114-8A, or rec114-8D strains in a dmc1Δ background; the region of the gel used for DSB quantification is indicated by brackets on the right side of the gel.
Figure 3.
Rec114 phosphorylation down-regulates Spo11 catalysis and Rec114-DSB hotspot association.
A. (i) Spo11-myc ChIP-chip profiles of REC114 (green), rec114-8A (red), and rec114-8D (blue) in a rad50S background for ChrIII. The centromere is denoted by a circle. For all ChIP-chip profiles presented in this work, ChIP/whole-cell extract (WCE) signal intensity was plotted against the chromosomal position after smoothing (bandwidth as indicated) and after decile normalization [17]. Brackets with stars label background peaks that become aligned among the profiles by this normalization. Cells were collected 6 hours after transfer to SPM, when the DSB level in a rad50S strain is near its maximum. (ii) qPCR results of ChIP of Spo11-myc in REC114, rec114-8A, and rec114-8D at the YCR047C DSB-hotspot located at position 211.7 kb on ChrIII [17]. B. (i) Rec114 –ChIP-chip profiles in REC114 (green), rec114-8A (red) and rec114-8D (blue) for ChrIII. Black bars: Hotspot positions [5]–[7]. (ii) qPCR time course of Rec114 -ChIP at a previously characterized axis site, located at 219.5 kb [17]. (iii, iv, v) Magnified views of a typical strong hotspot on ChrIV: REC114 (green), rec114-8A (red), rec114-8D (blue), Spo11-oligo counts from [7](black bars). In (v) all profiles were normalized by wild type, as an example for the mirror-like behavior of phospho-mimicking versus non-phosphorylatable Rec114 at DSB-hotspots. (vi) qPCR time course of Rec114-ChIP, at a hotspot (211,7 kb) and an axis site (219 kb) on ChrIII, expressed as ratio of hotspot/core to demonstrate that all three strains increase Rec114 hotspot occupancy relative to its axis binding as a function of time. Notably the extent of increase is greatest in rec114-8A, followed by REC114, and then rec114-8D. C. (i, ii) Genome wide correlation between DSB-hotspots and peaks of Spo11-myc and Rec114 ChIP-chip profiles: Both plots describe how well the 500 strongest peaks of a certain profile colocalize with the 500 strongest DSBs mapped by [7] (see also Method section). The cumulative fraction of peaks of a specified profile is plotted against the distance from the nearest DSB-cluster (in kb). For example, over 60% of Spo11-myc, rec114-8A/8D peaks are within 600 bp of one of the 500 strongest DSBs (600 bp distance marked with black line for convenience). A random model would predict only 7% of overlaps under these conditions. (i) Spo11-myc ChIP-chip profile analysis in the rad50S background: rec114-8A/rec114-8D (8A/8D), rec114-8A/REC114 (8A/WT), REC114/rec114-8A (WT/8A), random model and 2%, 98% percentiles (black). (ii) Rec114 ChIP-chip profile analysis in a RAD50 DMC1 background: Rec1148A/Rec1148D (8A/8D, red)), Rec114WT/Rec1148D (WT/8D), Rec1148A/Rec114WT (8A/WT), 1/Rec1148D (1/8D) Rec114/1 (WT), random model and 2%, 98% percentiles (black). For comparison, Spo11-myc in the rad50S background of rec114-8A/rec114-8D (8A/8D; bright green) is included.
Figure 4.
Rec114 phosphorylation delays its NDT80-dependent turnover.
A and B. Samples from indicated genotypes were collected at the specified time points and subjected to Western blot analysis using α-Rec114 or α-Hop1 antibodies. The graphs show the level of Rec114 in the Western blot, normalized to the total Hop1 signal (A) or to the loading control (B), and expressed relative to the t = 0 sample, set to 1. In ndt80Δ (B), the quantification of Rec1148D protein only shows timepoints 3, 6, and 12 hours.
Figure 5.
Effect of Rec114 phosphorylation on its synapsis dependent removal.
A. Temporal and spatial dynamics of Rec114 and Zip1 localization are assessed cytologically using antibodies against each protein. Presented are representative images of cells in leptotene/zygotene (i); zygotene/pachytene (ii); and pachytene (iii). The classification was based on the extent of Zip1-polymerization. White arrowheads: examples of the mutual exclusiveness of Rec114 and Zip1 signals. Scale bar: 5 µm. B. The fraction of REC114 ndt80Δ cells with Rec114 foci (black lines) or Zip1-linear stretches (orange lines). Grey columns; the average number of Rec114 foci per cell. C. (i) Fraction of Rec114-foci co-localizing with either Zip1-foci (yellow) or Zip1-lines (green). For each time point, ∼500 Rec114-foci collected from ∼ REC114 ndt80Δ nuclei were analyzed. (ii) Fraction of Zip1-lines co-localizing with Rec114-foci in the same ∼50 REC114 ndt80Δ nuclei per time point analyzed in panel (i). D. The average number of Rec114 foci (i), fraction of cells containing Rec114 foci (ii), and fraction of cells containing Zip1-linear stretches (iii) in REC114 ndt80Δ (green), rec114-8A ndt80Δ (red) or rec114-8D ndt80Δ (blue) cells.
Figure 6.
Model: Multiple mechanisms of regulating Rec114 contribute to meiotic DSB homeostasis.
A. Tel1/Mec1 phosphorylation of Rec114 following a successful Spo11-cleavage leads to local inhibition of DSB formation near the break. Given that most of Spo11-breaks are generated during leptotene, a feedback mechanism based on successful Spo11 catalysis would be most effective during this period, contributing to a large reduction in the DSB-catalyzing potential of the cell as depicted by A′. B. Synapsis-dependent Rec114-removal from chromosomes during the zygotene to pachytene transition contributes to a modest reduction in the DSB-catalyzing potential of the cell as depicted by B′. C. Ndt80-dependent Rec114-turnover would lead to irreversible inactivation of DSB-catalyzing potential at the genome-wide level (C′). The continued DSB formation observed in ndt80Δ strains [39] could be attributable to the persistent low level DSB catalyzing potential. D. Tel1/Mec1 activation of Hop1/Mek1 checkpoint function inhibits Ndt80, which in turn, ensures that cells do not progress through meiosis I until DSB repair is complete. Involvement of Ndt80 in Rec114 degradation (Figure 4) suggests that Tel1/Mec1, depending on circumstances, might also positive regulate DSB levels by preventing irreversible inactivation of DSB machinery.