Positive regulation of meiotic DNA double-strand break formation by activation of the DNA damage checkpoint kinase Mec1(ATR)

During meiosis, formation and repair of programmed DNA double-strand breaks (DSBs) create genetic exchange between homologous chromosomes-a process that is critical for reductional meiotic chromosome segregation and the production of genetically diverse sexually reproducing populations. Meiotic DSB formation is a complex process, requiring numerous proteins, of which Spo11 is the evolutionarily conserved catalytic subunit. Precisely how Spo11 and its accessory proteins function or are regulated is unclear. Here, we use Saccharomyces cerevisiae to reveal that meiotic DSB formation is modulated by the Mec1(ATR) branch of the DNA damage signalling cascade, promoting DSB formation when Spo11-mediated catalysis is compromised. Activation of the positive feedback pathway correlates with the formation of single-stranded DNA (ssDNA) recombination intermediates and activation of the downstream kinase, Mek1. We show that the requirement for checkpoint activation can be rescued by prolonging meiotic prophase by deleting the NDT80 transcription factor, and that even transient prophase arrest caused by Ndt80 depletion is sufficient to restore meiotic spore viability in checkpoint mutants. Our observations are unexpected given recent reports that the complementary kinase pathway Tel1(ATM) acts to inhibit DSB formation. We propose that such antagonistic regulation of DSB formation by Mec1 and Tel1 creates a regulatory mechanism, where the absolute frequency of DSBs is maintained at a level optimal for genetic exchange and efficient chromosome segregation

A nucleotide resolution map of Top2-linked DNA breaks in the yeast and human genome

DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage—distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo

Recombinase-independent chromosomal rearrangements between dispersed inverted repeats in Saccharomyces cerevisiae meiosis

DNA double-strand break (DSB) repair by homologous recombination (HR) uses a DNA template with similar sequence to restore genetic identity. Allelic DNA repair templates can be found on the sister chromatid or homologous chromosome. During meiotic recombination, DSBs preferentially repair from the homologous chromosome, with a proportion of HR events generating crossovers. Nevertheless, regions of similar DNA sequence exist throughout the genome, providing potential DNA repair templates. When DSB repair occurs at these non-allelic loci (termed ectopic recombination), chromosomal duplications, deletions and rearrangements can arise. Here, we characterize in detail ectopic recombination arising between a dispersed pair of inverted repeats in wild-type Saccharomyces cerevisiae at both a local and a chromosomal scale– the latter identified via gross chromosomal acentric and dicentric chromosome rearrangements. Mutation of the DNA damage checkpoint clamp loader Rad24 and the RecQ helicase Sgs1 causes an increase in ectopic recombination. Unexpectedly, additional mutation of the RecA orthologues Rad51 and Dmc1 alters––but does not abolish––the type of ectopic recombinants generated, revealing a novel class of inverted chromosomal rearrangement driven by the single-strand annealing pathway. These data provide important insights into the role of key DNA repair proteins in regulating DNA repair pathway and template choice during meiosis

Recombinase-independent chromosomal rearrangements between dispersed inverted repeats in Saccharomyces cerevisiae meiosis

DNA double-strand break (DSB) repair by homologous recombination (HR) uses a DNA template with similar sequence to restore genetic identity. Allelic DNA repair templates can be found on the sister chromatid or homologous chromosome. During meiotic recombination, DSBs preferentially repair from the homologous chromosome, with a proportion of HR events generating crossovers. Nevertheless, regions of similar DNA sequence exist throughout the genome, providing potential DNA repair templates. When DSB repair occurs at these non-allelic loci (termed ectopic recombination), chromosomal duplications, deletions and rearrangements can arise. Here, we characterize in detail ectopic recombination arising between a dispersed pair of inverted repeats in wild-type Saccharomyces cerevisiae at both a local and a chromosomal scale—the latter identified via gross chromosomal acentric and dicentric chromosome rearrangements. Mutation of the DNA damage checkpoint clamp loader Rad24 and the RecQ helicase Sgs1 causes an increase in ectopic recombination. Unexpectedly, additional mutation of the RecA orthologues Rad51 and Dmc1 alters—but does not abolish—the type of ectopic recombinants generated, revealing a novel class of inverted chromosomal rearrangement driven by the single-strand annealing pathway. These data provide important insights into the role of key DNA repair proteins in regulating DNA repair pathway and template choice during meiosis

Meiotic prophase length modulates Tel1-dependent DNA double-strand break interference

During meiosis, genetic recombination is initiated by the formation of many DNA double-strand breaks (DSBs) catalysed by the evolutionarily conserved topoisomerase-like enzyme, Spo11, in preferred genomic sites known as hotspots. DSB formation activates the Tel1/ATM DNA damage responsive (DDR) kinase, locally inhibiting Spo11 activity in adjacent hotspots via a process known as DSB interference. Intriguingly, in S. cerevisiae, over short genomic distances (</p

NDT80_Submission_Data_03.xlsx.

During meiosis, genetic recombination is initiated by the formation of many DNA double-strand breaks (DSBs) catalysed by the evolutionarily conserved topoisomerase-like enzyme, Spo11, in preferred genomic sites known as hotspots. DSB formation activates the Tel1/ATM DNA damage responsive (DDR) kinase, locally inhibiting Spo11 activity in adjacent hotspots via a process known as DSB interference. Intriguingly, in S. cerevisiae, over short genomic distances (NDT80 transcription factor. We propose that extension of meiotic prophase enables most cells, and therefore most chromosomal domains within them, to reach an equilibrium state of similar Spo11-DSB potential, reducing the impact that priming has on estimates of coincident DSB formation. Consistent with this view, when Tel1 is absent but Ndt80 is present and thus cells are able to rapidly exit meiotic prophase, genome-wide maps of Spo11-DSB formation are skewed towards pericentromeric regions and regions that load pro-DSB factors early—revealing regions of preferential priming—but this effect is abolished when NDT80 is deleted. Our work highlights how the stochastic nature of Spo11-DSB formation in individual cells within the limited temporal window of meiotic prophase can cause localised DSB clustering—a phenomenon that is exacerbated in tel1Δ cells due to the dual roles that Tel1 has in DSB interference and meiotic prophase checkpoint control.</div

Identification of Spo11 hotspots.

a, Diagram representing the hotspot calling method (see Extended method, “Hotspot identification”). The frequency of HpM was smoothed using a 201 bp Hann window with a minimum length of 25 bp, 25 reads and a cut-off of 0.193 HpM to filter for noise signal. Hotspots separated by sae2Δ ndt80Δ and sae2Δ ndt80Δ tel1Δ (Neale template). b–c, Venn diagrams of overlap between hotspots identified in this study by CC-seq (Neale) and hotspots identified by Spo11oligo mapping by Pan et al. 2011 [40] (b) or Mohibullah et al 2017 [60] (c). d–f, Distribution of hotspot frequency strengths for the total and unique hotspots identified by Neale vs Pan (d), Pan vs Neale (e) and Mohibullah vs Neale (f). g, Venn diagrams of overlap between hotspots identified in the Neale template and the non-specific hotspots identified in the spo11-Y135F strain. The cut-off for hotspot calling in the sae2Δ ndt80Δ spo11-Y135F mutant was lowered to 0.125 HpM. h, as in d–f but sae2Δ ndt80Δ spo11-Y135F vs Neale template. (TIFF)</p

Tel1-dependent genome-wide effect on DSB distribution.

a–c, Pearson correlation of Spo11 hotspot strengths (NormHpM) in the presence and absence of Tel1 in SAE2+ (Spo11-oligo maps; [60]) (a), and in CC-seq maps in sae2Δ (b) and sae2Δ ndt80Δ (c) strains. d, Visualization of the relative Spo11 hotspot intensities on chromosome IV in the indicated strains. e, Ratio of relative Spo11 hotspot intensities ±TEL1 on chromosome IV in SAE2+ cells (Spo11-oligo data; upper panel) and in CC-seq maps in the presence (middle panel) and absence (lower panel) of Ndt80. Values above zero indicate a higher DSB frequency in the presence of Tel1 and below zero a higher DSB frequency in the absence of Tel1. Fold change was smoothed to highlight the spatial trend caused by TEL1 deletion (black line). Other chromosomes are presented in S7 Fig. f–g, Heat maps of CC-seq data (sae2Δ) representing the ±Tel1 effect in the presence (f) and absence of Ndt80 (g). Log2 ratio of relative hotspot strengths ±TEL1 was binned into 50 kb intervals and plotted centred on the centromere and ranked by chromosome size. h, Pattern of Rec114 association time in hours as reported by Murakami et al (2020) [84] and presented as in f-g (reproduced from Fig 3H to aid visual comparison). i, Scatter plot of log2 fold change (TEL1/tel1Δ) ±NDT80 presented in (f) and (g) against Rec114 association time (h) for each 50 kb bin. The plotting order of the Rec114 data is reversed to match Fig 3I.</p

Meiotic prophase length homogenises the potential of forming active domains in which DSB formation may arise at short range.

a, Schematic representation of a heterogeneous mixture of cells with active and inactive domains with differing potential for DSB formation in NDT80+ cells. The formation of such active/inactive subdomains will bias the measurement of DSB frequency leading to underestimates of DSB interference. In the presence of Tel1, underestimation of the coincident DSB probability within the active domains would generate what appears to be weaker interference than expected, whereas, in the absence of Tel1 (tel1Δ), the lack of local DSB inhibition will enable coincident cutting (DSB clustering) in the fraction of cells with the active domain, causing negative interference. In this example we represent a situation in which 50% of the assayed population have the domain pre-activated at the tested region. b, We propose that deletion of NDT80 extends the length of the meiotic prophase homogenising the potential for domains to be pre-activated and allowing a more accurate estimate of DSB frequency per active domain. In the presence of Tel1, localised inhibition will cause DSBs to arise more evenly across the genome—leading to detection of positive interference, whereas in tel1Δ cells, the lack of inhibition will lead to detection of no interference. In this example we represent a situation in which 100% of the assayed population have the domain pre-activated at the tested region. Although Spo11-DSB formation arises in the context of a maturing loop-axis chromosome structure organised by cohesin, and contains chromatin loops that are within the size range (in S. cerevisiae) over which we infer pre-activation to occur (discussion for further details).</p

Deletion of <i>NDT80</i> influences the distribution of DSBs at a genome-wide scale.

a, Schematic of the genome-wide CC-seq Spo11-DSB mapping technique (see Extended methods). b–c, Pearson correlation of Spo11 hotspot strengths (NormHpM) in the presence and absence of Ndt80 in TEL1+ (b) and tel1Δ cells (c). d, Visualization of the relative Spo11 hotspot intensities on chromosome IV in the indicated strains. e, Ratio of relative Spo11 hotspot intensities ±NDT80 on chromosome IV in the presence (upper panel) and absence (lower panel) of Tel1. Values above zero indicate a higher DSB frequency in the presence of Ndt80 and below zero a higher DSB frequency in the absence of Ndt80. Fold change was smoothed to highlight the spatial trend effect of NDT80 deletion (black line). Other chromosomes are presented in S6 Fig. f–g, Heat maps representing ±Ndt80 effect in the presence (f) and absence of Tel1 (g). Log2 ratio of relative hotspot strengths ±NDT80 was binned into 50 kb intervals and plotted centred at the centromere and ranked by chromosome size. h, Pattern of Rec114 association time in hours as reported by Murakami et al (2020) [84] and presented as in f-g. i, Scatter plot of log2 fold change (NDT80/ndt80Δ) ±TEL1 presented in (f) and (g) against Rec114 association time (h) for each 50 kb bin. The plotting order of the Rec114 data is reversed to visualise the positive relationship between early Rec114 association and regions that are enhanced in the presence of NDT80, an effect that is stronger in the absence of Tel1.</p