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 protein, 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, Tel1(ATM). 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 Tel1(ATM) 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.

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.

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.

12DNA topoisomerases are required to resolve DNA topological stress. Despite this essential 13 role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, 14 jeopardising genome stability. Here, to understand the genomic distribution and 15 mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA 16 cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human 17 genomes-and use the meiotic Spo11 protein to validate the broad applicability of this 18 method to explore the role of diverse topoisomerase family members. Our data 19 characterises Mre11-dependent repair in yeast, and defines two strikingly different fractions 20 of Top2 activity in humans: tightly localised CTCF-proximal and broadly distributed 21 transcription-proximal. Moreover, the nucleotide resolution accuracy of our assay reveals 22 the influence primary DNA sequence has upon Top2 cleavage-for the first time 23 distinguishing canonical DNA double-strand breaks (DSBs) from a major population of DNA 24 single-strand breaks (SSBs) induced by etoposide (VP16) in vivo. 25 26 27 101 1995; Keeney et al., 1997), an orthologue of archaeal Topoisomerase VI (Bergerat et al., 102 1997). We first verified this enrichment principle using meiotic sae2Δ cells, in which Spo11-103 linked DSBs are known to accumulate at defined loci due to abrogation of the nucleolytic 104 pathway that releases Spo11 (Keeney and Kleckner, 1995; Neale et al., 2005). To 105 demonstrate specific enrichment of protein-linked molecules, total genomic DNA from 106 Gittens et al. 2019 meiotic S. cerevisiae cells was isolated in the absence of proteolysis, digested with PstI 107 restriction enzyme, and isolated on glass-fibre spin columns (Figure 1A, Methods). We 108 used eluted material to assay a known Spo11-DSB hotspot by Southern blotting (Figure 109 1B). While DSB fragments are a minor fraction of input material (~10% of total), and were 110 absent in wash fractions, DSBs accounted for >99% of total eluted material, indicating 111 ~1000-fold enrichment relative to non-protein-linked DNA (Figure 1B). 112 113 CC-seq maps known Spo11-DSB hotspots genome-wide with high reproducibility 114 To generate a genome-wide map of Spo11-DSBs, genomic DNA from meiotic sae2∆ cells 115 was sonicated to <400 bp in length, enriched upon the silica column, eluted, and ligated to 116 DNA adapters in a two-step procedure that utilised the known phosphotyrosyl-unlinking 117 activity of mammalian TDP2 to uncap the Spo11-bound end ('CC-seq'; Figure 1A / 118 Methods) (Cortes Ledesma et al., 2009; Johnson et al., 2019). Libraries were paired-end 119 sequenced and mapped to the S. cerevisiae reference genome (Table S1) alongside reads 120 from a previous mapping technique ('Spo11-oligo-seq') that relies on the isolation of 121 Spo11-linked oligonucleotides generated in wild-type cells during DSB repair (Pan et al., 122 2011). CC-seq revealed sharp, localised peaks ('hotspots') in SPO11+ cells (Figure 1C, 123 middle) that visually (Figu...

18 19 Meiotic recombination events are initiated by DNA double-strand breaks (DSBs) created by 20 the topoisomerase-like protein, Spo11. Similar to type-II topoisomerases, Spo11 becomes 21 covalently linked to the 5¢ ends generated on each side of the DSB. Whilst Spo11-oligos-the 22 *