Abstract
Meiotic recombination is a critical step in gametogenesis for many organisms, enabling the creation of genetically diverse haploid gametes. In each meiotic cell, recombination is initiated by numerous DNA double-strand breaks (DSBs) created by Spo11, the evolutionarily conserved topoisomerase-like protein1, but how these DSBs are distributed relatively uniformly across the four chromatids that make up each chromosome pair is poorly understood. Here we employ Saccharomyces cerevisiae to demonstrate distance-dependent DSB interference in cis (in which the occurrence of a DSB suppresses adjacent DSB formation)—a process that is mediated by the conserved DNA damage response kinase, Tel1ATM. The inhibitory function of Tel1 acts on a relatively local scale, while over large distances DSBs have a tendency to form independently of one another even in the presence of Tel1. Notably, over very short distances, loss of Tel1 activity causes DSBs to cluster within discrete zones of concerted DSB activity. Our observations support a hierarchical view of recombination initiation where Tel1ATM prevents clusters of DSBs, and further suppresses DSBs within the surrounding chromosomal region. Such collective negative regulation will help to ensure that recombination events are dispersed evenly and arranged optimally for genetic exchange and efficient chromosome segregation.
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Acknowledgements
V.G. was supported by an MRC New Investigator Grant to M.J.N. M.J.N. is supported by a University Research Fellowship from the Royal Society, a Career Development Award from the Human Frontiers Science Program Organisation, and a Consolidator Grant from the European Research Council. We thank S. Keeney and N. Mohibullah for sharing unpublished observations.
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V.G. and M.J.N. designed the experiments, analysed the data and wrote the paper. V.G., R.M.A., S.G. and M.J.N. performed the experiments. T.J.C. provided data analysis, mathematical modelling and bioinformatics support.
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Extended data figures and tables
Extended Data Figure 1 Tel1 suppresses the formation of multiple DSBs on the same chromatid.
a, Top: agarose-embedded genomic DNA isolated at the indicated time points was fractionated by PFGE, transferred to nylon membrane and hybridized with probes recognizing a central position on chromosome III, VIII, V and XI. Example lane profiles depict the relative signal density for the 8 h time points. Representative blots are shown. Areas defined for quantification of multi-cut DSBs (bottom panel) are indicated. Asterisk: cross-hybridization band. b, Quantification of total chromosome breakage measured in a. c, As in a but using probes specific to the left (top panel), or right (bottom panel) telomere. In agreement with more DSBs per chromatid being formed in the absence of Tel1, close inspection of the PFGE lane profiles revealed that dmc1Δ tel1Δ cells had an increased frequency of shorter chromosome fragments, yet also fewer large chromosome fragments. Because a similar shift in DSB distribution towards shorter molecules is also observed when chromosomes are probed from their opposite end (compare top and bottom panels), this apparent shift can be explained by an increase in the frequency of multiple DSBs arising on the same chromatid in dmc1Δ tel1Δ relative to dmc1Δ. a–c, Error bars, s.d. n = 3.
Extended Data Figure 2 Nonlinear increases in the frequency of closely spaced DSBs that arise upon TEL1 deletion cannot be explained by increases in absolute DSB frequency.
a–d, To test whether the nonlinear increase in double-cutting frequency for shorter molecules (Fig. 1c) could alternatively be explained by increases in DSB formation unassociated with any change in DSB interference, DSB formation on chromosome V (576 kb) was simulated 1 million times for each of the mean values of 2.5, 3, 3.5 and 4 DSBs per chromatid using DSB frequencies (per round of simulation) described by the Poisson distribution for the specified mean. These frequencies are approximately equivalent to 217, 260, 304 and 347 DSBs per cell (∼50 Mb). To simulate the frequency distributions of fragments detected by an interstitial probe, tallies were made of only those fragments that include the simulated probe position (FIR1 at position ∼220 kb). Subsequently, ratios were calculated for each position within each of these simulated distributions and equivalent simulated distributions generated with mean DSB frequencies 1.5× (red), 2× (green), 3× (purple), and 4× (blue) greater than the baseline. Finally, these data simulations were overlaid with the experimental observations made from chromosome V using the FIR1 probe when comparing the ratio of the dmc1Δ tel1Δ: dmc1Δ (data from Fig. 1c; orange). In all cases, as in Fig. 1c, data has been trimmed for fragments shorter than 50 kb and greater than 300 kb. The asterisks indicate instances of similarity between simulated and observed patterns. We note that in no circumstances do the simulations match the steep nonlinear curve, which is a hallmark of the experimental data caused by TEL1 deletion. The closest match is arguably simulating the ratio between a starting mean DSB frequency of 3.5 and that obtained from a 3–4-fold increase (c). While these simulations create a potential match, they both require the relatively high initial frequency of DSB formation in dmc1Δ cells of 304 DSBs per cell (note that the wild-type average frequency is estimated at ∼160 DSBs per cell1), increasing to 900–1,200 DSBs per cell upon TEL1 deletion. Moreover, in accord with the increased DSB frequency per cell, site-specific DSB frequencies would increase 3–4-fold in dmc1Δ tel1Δ cells relative to dmc1Δ cells to fit this simulation, something that we do not observe: average fold-changes in both sae2Δ and dmc1Δ strains are only ∼1.5× upon TEL1 deletion (Fig. 3c, Extended Data Fig. 4c and Extended Data Fig. 6a), a fold-change that is modelled by each of the red plots—all of which show very poor correlations with the observed data. Thus we conclude that the nonlinear inverse correlation between the fold-increase and the inter-DSB fragment length cannot solely arise from a global increase in DSB formation, but rather because the closer two DSBs are, the more likely that coincident cleavage is derepressed in the tel1Δ strain—as expected for a loss of cis-interference.
Extended Data Figure 3 Tel1-mediated DSB interference spans less than 150 kb.
a, Physical map of chromosome III showing relative position of DSB zones and probes. b, d, Agarose-embedded genomic DNA isolated at the indicated time points was fractionated by PFGE, transferred to nylon membrane and hybridized with probes recognizing a left (b), right (c) or central position (d) on chromosome III. Probes, main DSB sites and areas selected for quantification of DSBs arising in individual zones are indicated. e–g, Quantification of DSB formation in zone A (e), zone B (f) and double-cuts arising from DSBs occurring in both A and B on the same molecule (g). h, Comparison of observed zone A-B double-cuts (g) to expected zone A-B double-cuts (calculated from single cut frequencies measured in e, f). We observe no statistical difference between observed and expected values at any time point (t-test: P values all above 0.25 except dmc1Δ tel1Δ t = 10 h sample, 0.061). i, Calculated DSB interference between DSB zones A and B. b–i, Error bars, s.d. n = 3. See Supplementary Discussion for further details of this analysis.
Extended Data Figure 4 Analysis of DSB interference between HIS4::LEU2 and leu2::hisG.
a, b, Agarose-embedded genomic DNA isolated at the indicated time points was fractionated by PFGE, transferred to nylon membrane and hybridized with probes recognizing a, the FRM2 locus located between the HIS4::LEU2 and leu2::hisG DSB hotspots, and b, the CHA1 locus on the left telomere of chromosome III. Areas selected for quantification are indicated. c, Analysis of DSB interference between HIS4::LEU2 and leu2::hisG regions. The frequency of DSB formation within HIS4::LEU2 and leu2::hisG regions were measured in the various strains from PFGE using CHA1 (b) and FRM2 (a) probes, respectively, and the frequency of double-cuts were measured using the FRM2 probe (a). Total DSBs arising within the leu2::hisG region were calculated by summing double-cuts and leu2::hisG DSBs. Standard deviation indicates the variation between repeat analyses (n = 3 for all samples except rad24Δ dmc1Δ: n = 2). See notes below table for further details.
Extended Data Figure 5 Analysis of DSB double-cutting at various genomic loci.
a, b, Agarose-embedded genomic DNA isolated from the indicated time points and strains was fractionated by PFGE, transferred to nylon membrane and hybridized with various probes: FRM2 (a); POL5, DOT5, CTR86, YCR061W (b). a, Detection of double-cut (left panel) and quantification (right panel). Major double-cut band corresponding to coincident DSBs at HIS4::LEU2 and leu2::hisG is indicated with a star. b, Detection of double-cuts on different chromosomes following PFGE in strains fully (dmc1Δ exo1Δ and sae2Δ) or partially (exo1Δ) defective for DSB repair (top panel). Asterisks: tel1Δ-specific double-cut signals. Diagram depicts possible double-cuts (bottom panels).
Extended Data Figure 6 Analysis of DSB interference across the ARE1 region.
a, DSB interference was calculated in sae2Δ (top) and sae2Δ tel1Δ (bottom) using the following formula: 1–f(observed double-cuts)/f(expected double-cuts), where the expected double-cut values were calculated using two methods. Left, single-cut frequencies were measured by Southern-blot using a TAF2 probe (for DSB sites on the left of ARE1) or a PWP2 and RSC6 probe (for the right-hand side of ARE1; Extended Data Table 2). Right, calculations were made after converting the measured Spo11-oligo frequency1 at each DSB site to a % DSB ± s.d. value by using the measured DSB frequency at ARE1 in sae2Δ or sae2Δ tel1Δ for normalization (see Notes below table and Methods for further details). b, Chart of observed (column B) and expected (column F and Q) frequencies of double-cuts. Error bars, s.d. n = 2. P values, two-tailed t-test. Double-cut products that were present at a frequency that was statistically different from that for no interference (independence) were highlighted in a according to the type of interference present: red indicates positive DSB interference, blue indicates negative DSB interference (concerted DSB formation); in b the same statistical differences were indicated with open diamonds or asterisks, respectively.
Extended Data Figure 7 Tel1 suppresses concerted DSB formation within chromatin loop domains at numerous chromosomal loci.
a–i, DSB interference was calculated across three DSB hotspot regions located on three different chromosomes: chromosome III, BUD23–ARE1 to YCR061W–BUD31 (a–c); chromosome VIII, BRL1–PUT2 to SRB2–NCP1 (d–f); and chromosome IV, YDR186C–CCT6 to MSS116–REF2 (g–i). a, d, g, Upper panels, genomic DNA isolated from sae2Δ or sae2Δ tel1Δ strains at the indicated time points was fractionated by agarose electrophoresis, transferred to nylon membrane and hybridized with the indicated probes: YCRO61W (a), SRB2 (d), CCT6 (g). Lower panels, diagram of mean RMM binding profile14 overlaid with Spo11-DSB hotspot peaks1. Intervals between various detectable double-cut events are indicated below and specified with the letters A to D. Probes used for detecting double-cuts by Southern blotting are indicated. b, e, h, Chart of observed and expected double-cuts for each of the indicated intervals, calculated as an average (per repeat) across the 4–10 h time points. Expected double-cut frequencies for each interval were calculated by multiplying the DSB frequencies (average across 4–10 h) at the two sites. Single-cut frequencies were measured by Southern-blot (see Extended Data Table 2 and Methods for details). For some intervals (superscript with a “+”), due to no Southern DSB data being available at the minor DSB site, calculations were made using the normalized Spo11-oligo frequency1 at the minor DSB site (as was performed in Fig. 4 and described in Methods). Asterisks and open diamonds indicate significant negative and positive interference, respectively. c, f, i, DSB interference was calculated by the following formula: 1 – f(observed double-cuts)/f(expected double-cuts). Values above zero indicate positive DSB interference. Values below zero indicate negative DSB interference (concerted DSB formation). Conclusion: In addition to ARE1 (Fig. 4), at all three additional loci tested, concerted DSB formation is localized predominantly within a domain approximately demarcated by the RMM binding profile (see a, d and g, lower panels). Notably, coincident formation of two DSBs, one within the BUD23-ARE1 domain and one within the YCR061W-BUD31 domain, arise independently in sae2Δ tel1Δ despite coincident DSB formation within each interval displaying negative interference. In a, double-cuts in interval A were measured using the ARE1 probe (Fig. 4a). Asterisk in a upper panel denotes a band that is a mixture of two tel1Δ-dependent double-cuts, which owing to the relative location of the YCR061W probe and DSB sites cannot be unambiguously assigned and therefore were not analysed. Error bars, s.d. n = 2, except g–i where only one experiment was performed. P values, two-tailed t-test.
Extended Data Figure 8 Stochastic loop tethering (activation) predicts apparent short-range negative interference.
a, In this model, DSBs A and B reside within a single loop domain (subject to tethering-dependent DSB formation), which is active in only a subpopulation of cells. The expected frequency of coincident DSB formation (double-cutting), assuming no DSB interference, is calculated for different frequencies of loop activation/tethering per chromatid assuming a model where DSB formation is wholly dependent on loop activation/tethering. In summary, loop activation/tethering at a frequency of X, will result in apparent negative interference of 1 – 1/X. See text for further details. b, c, Cartoons (left) and worked examples (right) for situations in which 50% (b) or 20% (c) of the chromatids within the assayed population are active/tethered at the test locus. The cartoons depict the tethering state of an average sample of 10 chromatids from the population. It is also possible that loop tethering and loop activation are not synonymous processes. In principle, activation of a loop might precede and enable tethering, but not be caused by it.
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Garcia, V., Gray, S., Allison, R. et al. Tel1ATM-mediated interference suppresses clustered meiotic double-strand-break formation. Nature 520, 114–118 (2015). https://doi.org/10.1038/nature13993
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DOI: https://doi.org/10.1038/nature13993
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