Meiotic DNA Double-strand Breaks Formation Research Articles

The TOPOVIBL meiotic DSB formation protein: new insights from its biochemical and structural characterization.

The TOPOVIL complex catalyzes the formation of DNA double strand breaks (DSB) that initiate meiotic homologous recombination, an essential step for chromosome segregation and genetic diversity during gamete production. TOPOVIL is composed of two subunits (SPO11 and TOPOVIBL) and is evolutionarily related to the archaeal TopoVI topoisomerase complex. SPO11 is the TopoVIA subunit orthologue and carries the DSB formation catalytic activity. TOPOVIBL shares homology with the TopoVIB ATPase subunit. TOPOVIBL is essential for meiotic DSB formation, but its molecular function remains elusive, partly due to the lack of biochemical studies. Here, we purified TOPOVIBLΔC25 and characterized its structure and mode of action in vitro. Our structural analysis revealed that TOPOVIBLΔC25 adopts a dynamic conformation in solution and our biochemical study showed that the protein remains monomeric upon incubation with ATP, which correlates with the absence of ATP binding. Moreover, TOPOVIBLΔC25 interacted with DNA, with a preference for some geometries, suggesting that TOPOVIBL senses specific DNA architectures. Altogether, our study identified specific TOPOVIBL features that might help to explain how TOPOVIL function evolved toward a DSB formation activity in meiosis.

Chromatin binding by HORMAD proteins regulates meiotic recombination initiation

The meiotic chromosome axis coordinates chromosome organization and interhomolog recombination in meiotic prophase and is essential for fertility. In S. cerevisiae, the HORMAD protein Hop1 mediates the enrichment of axis proteins at nucleosome-rich islands through a central chromatin-binding region (CBR). Here, we use cryoelectron microscopy to show that the Hop1 CBR directly recognizes bent nucleosomal DNA through a composite interface in its PHD and winged helix–turn–helix domains. Targeted disruption of the Hop1 CBR-nucleosome interface causes a localized reduction of axis protein binding and meiotic DNA double-strand breaks (DSBs) in axis islands and leads to defects in chromosome synapsis. Synthetic effects with mutants of the Hop1 regulator Pch2 suggest that nucleosome binding delays a conformational switch in Hop1 from a DSB-promoting, Pch2-inaccessible state to a DSB-inactive, Pch2-accessible state to regulate the extent of meiotic DSB formation. Phylogenetic analyses of meiotic HORMADs reveal an ancient origin of the CBR, suggesting that the mechanisms we uncover are broadly conserved.

A bi-allelic REC114 loss-of-function variant causes meiotic arrest and nonobstructive azoospermia.

Nonobstructive azoospermia (NOA), the most severe manifestation of male infertility, lacks a comprehensive understanding of its genetic etiology. Here, a bi-allelic loss-of-function variant in REC114 (c.568C > T: p.Gln190*) were identified through whole exome sequencing (WES) in a Chinese NOA patient. Testicular histopathological analysis and meiotic chromosomal spread analysis were conducted to assess the stage of spermatogenesis arrested. Co-immunoprecipitation (Co-IP) and Western blot (WB) were used to investigate the influence of variant in vitro. In addition, our results revealed that the variant resulted in truncated REC114 protein and impaired interaction with MEI4, which was essential for meiotic DNA double-strand break (DSB) formation. As far as we know, this study presents the first report that identifies REC114 as the causative gene for male infertility. Furthermore, our study demonstrated indispensability of the REC114-MEI4 complex in maintaining DSB homoeostasis, and highlighted that the disruption of the complex due to the REC114 variant may underline the mechanism of NOA.

FIGNL1 AAA+ ATPase remodels RAD51 and DMC1 filaments in pre-meiotic DNA replication and meiotic recombination

The formation of RAD51/DMC1 filaments on single-stranded (ss)DNAs essential for homology search and strand exchange in DNA double-strand break (DSB) repair is tightly regulated. FIGNL1 AAA+++ ATPase controls RAD51-mediated recombination in human cells. However, its role in gametogenesis remains unsolved. Here, we characterized a germ line-specific conditional knockout (cKO) mouse of FIGNL1. Fignl1 cKO male mice showed defective chromosome synapsis and impaired meiotic DSB repair with the accumulation of RAD51/DMC1 on meiotic chromosomes, supporting a positive role of FIGNL1 in homologous recombination at a post-assembly stage of RAD51/DMC1 filaments. Fignl1 cKO spermatocytes also accumulate RAD51/DMC1 on chromosomes in pre-meiotic S-phase. These RAD51/DMC1 assemblies are independent of meiotic DSB formation. We also showed that purified FIGNL1 dismantles RAD51 filament on double-stranded (ds)DNA as well as ssDNA. These results suggest an additional role of FIGNL1 in limiting the non-productive assembly of RAD51/DMC1 on native dsDNAs during pre-meiotic S-phase and meiotic prophase I.

Structure and DNA-bridging activity of the essential Rec114-Mei4 trimer interface.

The DNA double-strand breaks (DSBs) that initiate meiotic recombination are formed by an evolutionarily conserved suite of factors that includes Rec114 and Mei4 (RM), which regulate DSB formation both spatially and temporally. In vivo, these proteins form large immunostaining foci that are integrated with higher-order chromosome structures. In vitro, they form a 2:1 heterotrimeric complex that binds cooperatively to DNA to form large, dynamic condensates. However, understanding of the atomic structures and dynamic DNA binding properties of RM complexes is lacking. Here, we report a structural model of a heterotrimeric complex of the C terminus of Rec114 with the N terminus of Mei4, supported by nuclear magnetic resonance experiments. This minimal complex, which lacks the predicted intrinsically disordered region of Rec114, is sufficient to bind DNA and form condensates. Single-molecule experiments reveal that the minimal complex can bridge two or more DNA duplexes and can generate force to condense DNA through long-range interactions. AlphaFold2 predicts similar structural models for RM orthologs across diverse taxa despite their low degree of sequence similarity. These findings provide insight into the conserved networks of protein-protein and protein-DNA interactions that enable condensate formation and promote formation of meiotic DSBs.

Differentiated function and localisation of SPO11-1 and PRD3 on the chromosome axis during meiotic DSB formation in Arabidopsis thaliana.

During meiosis, DNA double-strand breaks (DSBs) occur throughout the genome, a subset of which are repaired to form reciprocal crossovers between chromosomes. Crossovers are essential to ensure balanced chromosome segregation and to create new combinations of genetic variation. Meiotic DSBs are formed by a topoisomerase-VI-like complex, containing catalytic (e.g. SPO11) proteins and auxiliary (e.g. PRD3) proteins. Meiotic DSBs are formed in chromatin loops tethered to a linear chromosome axis, but the interrelationship between DSB-promoting factors and the axis is not fully understood. Here, we study the localisation of SPO11-1 and PRD3 during meiosis, and investigate their respective functions in relation to the chromosome axis. Using immunocytogenetics, we observed that the localisation of SPO11-1 overlaps relatively weakly with the chromosome axis and RAD51, a marker of meiotic DSBs, and that SPO11-1 recruitment to chromatin is genetically independent of the axis. In contrast, PRD3 localisation correlates more strongly with RAD51 and the chromosome axis. This indicates that PRD3 likely forms a functional link between SPO11-1 and the chromosome axis to promote meiotic DSB formation. We also uncovered a new function of SPO11-1 in the nucleation of the synaptonemal complex protein ZYP1. We demonstrate that chromosome co-alignment associated with ZYP1 deposition can occur in the absence of DSBs, and is dependent on SPO11-1, but not PRD3. Lastly, we show that the progression of meiosis is influenced by the presence of aberrant chromosomal connections, but not by the absence of DSBs or synapsis. Altogether, our study provides mechanistic insights into the control of meiotic DSB formation and reveals diverse functional interactions between SPO11-1, PRD3 and the chromosome axis.

Multi-color dSTORM microscopy in Hormad1-/- spermatocytes reveals alterations in meiotic recombination intermediates and synaptonemal complex structure.

Recombinases RAD51 and its meiosis-specific paralog DMC1 accumulate on single-stranded DNA (ssDNA) of programmed DNA double strand breaks (DSBs) in meiosis. Here we used three-color dSTORM microscopy, and a mouse model with severe defects in meiotic DSB formation and synapsis (Hormad1-/-) to obtain more insight in the recombinase accumulation patterns in relation to repair progression. First, we used the known reduction in meiotic DSB frequency in Hormad1-/- spermatocytes to be able to conclude that the RAD51/DMC1 nanofoci that preferentially localize at distances of ~300 nm form within a single DSB site, whereas a second preferred distance of ~900 nm, observed only in wild type, represents inter-DSB distance. Next, we asked whether the proposed role of HORMAD1 in repair inhibition affects the RAD51/DMC1 accumulation patterns. We observed that the two most frequent recombinase configurations (1 DMC1 and 1 RAD51 nanofocus (D1R1), and D2R1) display coupled frequency dynamics over time in wild type, but were constant in the Hormad1-/- model, indicating that the lifetime of these intermediates was altered. Recombinase nanofoci were also smaller in Hormad1-/- spermatocytes, consistent with changes in ssDNA length or protein accumulation. Furthermore, we established that upon synapsis, recombinase nanofoci localized closer to the synaptonemal complex (SYCP3), in both wild type and Hormad1-/- spermatocytes. Finally, the data also revealed a hitherto unknown function of HORMAD1 in inhibiting coil formation in the synaptonemal complex. SPO11 plays a similar but weaker role in coiling and SYCP1 had the opposite effect. Using this large super-resolution dataset, we propose models with the D1R1 configuration representing one DSB end containing recombinases, and the other end bound by other ssDNA binding proteins, or both ends loaded by the two recombinases, but in below-resolution proximity. This may then often evolve into D2R1, then D1R2, and finally back to D1R1, when DNA synthesis has commenced.

The meiotic topoisomerase VI B subunit (MTOPVIB) is essential for meiotic DNA double-strand break formation in barley (Hordeum vulgare L.)

Key messageIn barley (Hordeum vulgare), MTOPVIB is critical for meiotic DSB and accompanied SC and CO formation while dispensable for meiotic bipolar spindle formation.Homologous recombination during meiosis assures genetic variation in offspring. Programmed meiotic DNA double-strand breaks (DSBs) are repaired as crossover (CO) or non-crossover (NCO) during meiotic recombination. The meiotic topoisomerase VI (TopoVI) B subunit (MTOPVIB) plays an essential role in meiotic DSB formation critical for CO-recombination. More recently MTOPVIB has been also shown to play a role in meiotic bipolar spindle formation in rice and maize. Here, we describe a meiotic DSB-defective mutant in barley (Hordeum vulgare L.). CRISPR-associated 9 (Cas9) endonuclease-generated mtopVIB plants show complete sterility due to the absence of meiotic DSB, synaptonemal complex (SC), and CO formation leading to the occurrence of univalents and their unbalanced segregation into aneuploid gametes. In HvmtopVIB plants, we also frequently found the bi-orientation of sister kinetochores in univalents during metaphase I and the precocious separation of sister chromatids during anaphase I. Moreover, the near absence of polyads after meiosis II, suggests that despite being critical for meiotic DSB formation in barley, MTOPVIB seems not to be strictly required for meiotic bipolar spindle formation.

Conservation and divergence of meiotic DNA double strand break forming mechanisms in Arabidopsis thaliana.

In the current meiotic recombination initiation model, the SPO11 catalytic subunits associate with MTOPVIB to form a Topoisomerase VI-like complex that generates DNA double strand breaks (DSBs). Four additional proteins, PRD1/AtMEI1, PRD2/AtMEI4, PRD3/AtMER2 and the plant specific DFO are required for meiotic DSB formation. Here we show that (i) MTOPVIB and PRD1 provide the link between the catalytic sub-complex and the other DSB proteins, (ii) PRD3/AtMER2, while localized to the axis, does not assemble a canonical pre-DSB complex but establishes a direct link between the DSB-forming and resection machineries, (iii) DFO controls MTOPVIB foci formation and is part of a divergent RMM-like complex including PHS1/AtREC114 and PRD2/AtMEI4 but not PRD3/AtMER2, (iv) PHS1/AtREC114 is absolutely unnecessary for DSB formation despite having a conserved position within the DSB protein network and (v) MTOPVIB and PRD2/AtMEI4 interact directly with chromosome axis proteins to anchor the meiotic DSB machinery to the axis.

Mechanism and Control of Meiotic DNA Double-Strand Break Formation in S. cerevisiae.

Developmentally programmed formation of DNA double-strand breaks (DSBs) by Spo11 initiates a recombination mechanism that promotes synapsis and the subsequent segregation of homologous chromosomes during meiosis. Although DSBs are induced to high levels in meiosis, their formation and repair are tightly regulated to minimize potentially dangerous consequences for genomic integrity. In S. cerevisiae, nine proteins participate with Spo11 in DSB formation, but their molecular functions have been challenging to define. Here, we describe our current view of the mechanism of meiotic DSB formation based on recent advances in the characterization of the structure and function of DSB proteins and discuss regulatory pathways in the light of recent models.

How do small chromosomes know they are small? Maximizing meiotic break formation on the shortest yeast chromosomes.

The programmed formation of DNA double-strand breaks (DSBs) in meiotic prophase I initiates the homologous recombination process that yields crossovers between homologous chromosomes, a prerequisite to accurately segregating chromosomes during meiosis I (MI). In the budding yeast Saccharomyces cerevisiae, proteins required for meiotic DSB formation (DSB proteins) accumulate to higher levels specifically on short chromosomes to ensure that these chromosomes make DSBs. We previously demonstrated that as-yet undefined cis-acting elements preferentially recruit DSB proteins and promote higher levels of DSBs and recombination and that these intrinsic features are subject to selection pressure to maintain the hyperrecombinogenic properties of short chromosomes. Thus, this targeted boosting of DSB protein binding may be an evolutionarily recurrent strategy to mitigate the risk of meiotic mis-segregation caused by karyotypic constraints. However, the underlining mechanisms are still elusive. Here, we discuss possible scenarios in which components of the meiotic chromosome axis (Red1 and Hop1) bind to intrinsic features independent of the meiosis-specific cohesin subunit Rec8 and DNA replication, promoting preferential binding of DSB proteins to short chromosomes. We also propose a model where chromosome position in the nucleus, influenced by centromeres, promotes the short-chromosome boost of DSB proteins.

ATM controls meiotic DNA double-strand break formation and recombination and affects synaptonemal complex organization in plants.

Meiosis is a specialized cell division that gives rise to genetically distinct gametic cells. Meiosis relies on the tightly controlled formation of DNA double-strand breaks (DSBs) and their repair via homologous recombination for correct chromosome segregation. Like all forms of DNA damage, meiotic DSBs are potentially harmful and their formation activates an elaborate response to inhibit excessive DNA break formation and ensure successful repair. Previous studies established the protein kinase ATM as a DSB sensor and meiotic regulator in several organisms. Here we show that Arabidopsis ATM acts at multiple steps during DSB formation and processing, as well as crossover (CO) formation and synaptonemal complex (SC) organization, all vital for the successful completion of meiosis. We developed a single-molecule approach to quantify meiotic breaks and determined that ATM is essential to limit the number of meiotic DSBs. Local and genome-wide recombination screens showed that ATM restricts the number of interference-insensitive COs, while super-resolution STED nanoscopy of meiotic chromosomes revealed that the kinase affects chromatin loop size and SC length and width. Our study extends our understanding of how ATM functions during plant meiosis and establishes it as an integral factor of the meiotic program.

A TOP6BL mutation abolishes meiotic DNA double-strand break formation and causes human infertility

Meiosis is pivotal for sexual reproduction and fertility. Meiotic programmed DNA double-strand breaks (DSBs) initiate homologous recombination, ensuring faithful chromosome segregation and generation of gametes. However, few studies have focused on meiotic DSB formation in human reproduction. Here, we report four infertile siblings born to a consanguineous marriage, with three brothers suffering from non-obstructive azoospermia and one sister suffering from unexplained infertility with normal menstrual cycles and normal ovary sizes with follicular activity. An autosomal recessive mutation in TOP6BL was found co-segregating with infertility in this family. Investigation of one male patient revealed failure in programmed meiotic DSB formation and meiotic arrest prior to pachytene stage of prophase I. Mouse models carrying similar mutations to that in patients recapitulated the spermatogenic abnormalities of the patient. Pathogenicity of the mutation in the female patient was supported by observations in mice that meiotic programmed DSBs failed to form in mutant oocytes and oocyte maturation failure due to absence of meiotic recombination. Our study thus illustrates the phenotypical characteristics and the genotype-phenotype correlations of meiotic DSB formation failure in humans.

Sharing Marks: H3K4 Methylation and H2B Ubiquitination as Features of Meiotic Recombination and Transcription.

Meiosis is a specialized cell division that gives raise to four haploid gametes from a single diploid cell. During meiosis, homologous recombination is crucial to ensure genetic diversity and guarantee accurate chromosome segregation. Both the formation of programmed meiotic DNA double-strand breaks (DSBs) and their repair using homologous chromosomes are essential and highly regulated pathways. Similar to other processes that take place in the context of chromatin, histone posttranslational modifications (PTMs) constitute one of the major mechanisms to regulate meiotic recombination. In this review, we focus on specific PTMs occurring in histone tails as driving forces of different molecular events, including meiotic recombination and transcription. In particular, we concentrate on the influence of H3K4me3, H2BK123ub, and their corresponding molecular machineries that write, read, and erase these histone marks. The Spp1 subunit within the Complex of Proteins Associated with Set1 (COMPASS) is a critical regulator of H3K4me3-dependent meiotic DSB formation. On the other hand, the PAF1c (RNA polymerase II associated factor 1 complex) drives the ubiquitination of H2BK123 by Rad6-Bre1. We also discuss emerging evidence obtained by cryo-electron microscopy (EM) structure determination that has provided new insights into how the “cross-talk” between these two marks is accomplished.

Advances Towards How Meiotic Recombination Is Initiated: A Comparative View and Perspectives for Plant Meiosis Research.

Meiosis is an essential cell-division process for ensuring genetic diversity across generations. Meiotic recombination ensures the accuracy of genetic interchange between homolous chromosomes and segregation of parental alleles. Programmed DNA double-strand breaks (DSBs), catalyzed by the evolutionarily conserved topoisomerase VIA (a subunit of the archaeal type II DNA topoisomerase)-like enzyme Spo11 and several other factors, is a distinctive feature of meiotic recombination initiation. The meiotic DSB formation and its regulatory mechanisms are similar among species, but certain aspects are distinct. In this review, we introduced the cumulative knowledge of the plant proteins crucial for meiotic DSB formation and technical advances in DSB detection. We also summarized the genome-wide DSB hotspot profiles for different model organisms. Moreover, we highlighted the classical views and recent advances in our knowledge of the regulatory mechanisms that ensure the fidelity of DSB formation, such as multifaceted kinase-mediated phosphorylation and the consequent high-dimensional changes in chromosome structure. We provided an overview of recent findings concerning DSB formation, distribution and regulation, all of which will help us to determine whether meiotic DSB formation is evolutionarily conserved or varies between plants and other organisms.

Conserved HORMA domain-containing protein Hop1 stabilizes interaction between proteins of meiotic DNA break hotspots and chromosome axis.

HORMA domain-containing proteins such as Hop1 play crucial regulatory roles in various chromosomal functions. Here, we investigated roles of the fission yeast Hop1 in the formation of recombination-initiating meiotic DNA double strand breaks (DSBs). Meiotic DSB formation in fission yeast relies on multiple protein-protein interactions such as the one between the chromosome axial protein Rec10 and the DSB-forming complex subunit Rec15. Chromatin immunoprecipitation sequencing demonstrated that Hop1 is colocalized with both Rec10 and Rec15, and we observed physical interactions of Hop1 to Rec15 and Rec10. These results suggest that Hop1 promotes DSB formation by interacting with both axis components and the DSB-forming complex. We also show that Hop1 binding to DSB hotspots requires Rec15 and Rec10, while Hop1 axis binding requires Rec10 only, suggesting that Hop1 is recruited to the axis via Rec10, and to hotspots by hotspot-bound Rec15. Furthermore, we introduced separation-of-function Rec10 mutations, deficient for interaction with either Rec15 or Hop1. These single mutations and hop1Δ conferred only partial defects in meiotic recombination, while the combining the Rec15-binding-deficient rec10 mutation with hop1Δ synergistically reduced meiotic recombination, at least at a model hotspot. Taken together, Hop1 likely functions as a stabilizer for Rec15–Rec10 interaction to promote DSB formation.

CDK contribution to DSB formation and recombination in fission yeast meiosis.

CDKs (cyclin-dependent kinases) associate with different cyclins to form different CDK-complexes that are fundamental for an ordered cell cycle progression, and the coordination of this progression with different aspects of the cellular physiology. During meiosis programmed DNA double-strand breaks (DSBs) initiate recombination that in addition to generating genetic variability are essential for the reductional chromosome segregation during the first meiotic division, and therefore for genome stability and viability of the gametes. However, how meiotic progression and DSB formation are coordinated, and the role CDKs have in the process, is not well understood. We have used single and double cyclin deletion mutants, and chemical inhibition of global CDK activity using the cdc2-asM17 allele, to address the requirement of CDK activity for DSB formation and recombination in fission yeast. We report that several cyclins (Cig1, Cig2, and the meiosis-specific Crs1) control DSB formation and recombination, with a major contribution of Crs1. Moreover, complementation analysis indicates specificity at least for this cyclin, suggesting that different CDK complexes might act in different pathways to promote recombination. Down-regulation of CDK activity impinges on the formation of linear elements (LinEs, protein complexes required for break formation at most DSB hotspot sites). This defect correlates with a reduction in the capability of one structural component (Rec25) to bind chromatin, suggesting a molecular mechanism by which CDK controls break formation. However, reduction in DSB formation in cyclin deletion mutants does not always correspondingly correlate with a proportional reduction in meiotic recombination (crossovers), suggesting that specific CDK complexes might also control downstream events balancing repair pathways. Therefore, our work points to CDK regulation of DSB formation as a key conserved feature in the initiation of meiotic recombination, in addition to provide a view of possible roles CDK might have in other steps of the recombination process.

Narya, a RING finger domain-containing protein, is required for meiotic DNA double-strand break formation and crossover maturation in Drosophila melanogaster.

Meiotic recombination, which is necessary to ensure that homologous chromosomes segregate properly, begins with the induction of meiotic DNA double-strand breaks (DSBs) and ends with the repair of a subset of those breaks into crossovers. Here we investigate the roles of two paralogous genes, CG12200 and CG31053, which we have named Narya and Nenya, respectively, due to their relationship with a structurally similar protein named Vilya. We find that narya recently evolved from nenya by a gene duplication event, and we show that these two RING finger domain-containing proteins are functionally redundant with respect to a critical role in DSB formation. Narya colocalizes with Vilya foci, which are known to define recombination nodules, or sites of crossover formation. A separation-of-function allele of narya retains the capacity for DSB formation but cannot mature those DSBs into crossovers. We further provide data on the physical interaction of Narya, Nenya and Vilya, as assayed by the yeast two-hybrid system. Together these data support the view that all three RING finger domain-containing proteins function in the formation of meiotic DNA DSBs and in the process of crossing over.

Mouse REC114 is essential for meiotic DNA double-strand break formation and forms a complex with MEI4.

Programmed formation of DNA double-strand breaks (DSBs) initiates the meiotic homologous recombination pathway. This pathway is essential for proper chromosome segregation at the first meiotic division and fertility. Meiotic DSBs are catalyzed by Spo11. Several other proteins are essential for meiotic DSB formation, including three evolutionarily conserved proteins first identified in Saccharomyces cerevisiae (Mer2, Mei4, and Rec114). These three S. cerevisiae proteins and their mouse orthologs (IHO1, MEI4, and REC114) co-localize on the axes of meiotic chromosomes, and mouse IHO1 and MEI4 are essential for meiotic DSB formation. Here, we show that mouse Rec114 is required for meiotic DSB formation. Moreover, MEI4 forms a complex with REC114 and IHO1 in mouse spermatocytes, consistent with cytological observations. We then demonstrated in vitro the formation of a stable complex between REC114 C-terminal domain and MEI4 N-terminal domain. We further determine the structure of the REC114 N-terminal domain that revealed similarity with Pleckstrin homology domains. These analyses provide direct insights into the architecture of these essential components of the meiotic DSB machinery.

A chromatin-associated protein required for inducing and limiting meiotic DNA double-strand break formation.

Programmed DNA double-strand breaks (DSBs) are required for meiotic recombination, but the number is strictly controlled because they are potentially harmful. Here we report a novel protein, Pars11, which is required for Spo11-dependent DSB formation in the protist Tetrahymena. Pars11 localizes to chromatin early in meiotic prophase in a Spo11-independent manner and is removed before the end of prophase. Pars11 removal depends on DSB formation and ATR-dependent phosphorylation. In the absence of the DNA damage sensor kinase ATR, Pars11 is retained on chromatin and excess DSBs are generated. Similar levels of Pars11 persistence and DSB overproduction occur in a non-phosphorylatable pars11 mutant. We conclude that Pars11 supports DSB formation by Spo11 until enough DSBs are formed; thereafter, DSB production stops in response to ATR-dependent degradation of Pars11 or its removal from chromatin. A similar DSB control mechanism involving a Rec114-Tel1/ATM-dependent negative feedback loop regulates DSB formation in budding yeast. However, there is no detectable sequence homology between Pars11 and Rec114, and DSB numbers are more tightly controlled by Pars11 than by Rec114. The discovery of this mechanism for DSB regulation in the evolutionarily distant protist and fungal lineages suggests that it is conserved across eukaryotes.