Fast and Precise: How to Measure Meiotic Crossovers in Arabidopsis

The FK506 Binding Protein Fpr3 Counteracts Protein Phosphatase 1 to Maintain Meiotic Recombination Checkpoint Activity

The eukaryotic recombinases RAD51 and DMC1 are essential for DNA strand-exchange between homologous chromosomes during meiosis. RAD51 is also expressed during mitosis, and mediates homologous recombination (HR) between sister chromatids. It has been suggested that DMC1 might be involved in the switch from intersister chromatid recombination in somatic cells to interhomolog meiotic recombination. At meiosis, the Arabidopsis Atrad51 null mutant fails to synapse and has extensive chromosome fragmentation. The Atdmc1 null mutant is also asynaptic, but in this case chromosome fragmentation is absent. Thus in plants, AtDMC1 appears to be indispensable for interhomolog homologous recombination, whereas AtRAD51 seems to be more involved in intersister recombination. In this work, we have studied a new AtRAD51 knock-down mutant, Atrad51-2, which expresses only a small quantity of RAD51 protein. Atrad51-2 mutant plants are sterile and hypersensitive to DNA double-strand break induction, but their vegetative development is apparently normal. The meiotic phenotype of the mutant consists of partial synapsis, an elevated frequency of univalents, a low incidence of chromosome fragmentation and multivalent chromosome associations. Surprisingly, non-homologous chromosomes are involved in 51% of bivalents. The depletion of AtDMC1 in the Atrad51-2 background results in the loss of bivalents and in an increase of chromosome fragmentation. Our results suggest that a critical level of AtRAD51 is required to ensure the fidelity of HR during interchromosomal exchanges. Assuming the existence of asymmetrical DNA strand invasion during the initial steps of recombination, we have developed a working model in which the initial step of strand invasion is mediated by AtDMC1, with AtRAD51 required to check the fidelity of this process.

Misincorporation of genomic uracil and formation of DNA double strand breaks are known consequences of exposure to TS inhibitors such as 5-fluorouracil, and pemetrexed. Uracil DNA glycosylase catalyzes the excision of genomic uracil and initiates DNA base excision repair (BER). Thus, a relationship between antifolate cytotoxicity and UNG expression and activity has been hypothesized. However, a precise mechanism linking antifolate-induced formation of DSBs to genomic uracil accumulation and UNG-initiated BER has not been described. Herein, we report that despite equivalent proliferation indices, DLD1 UNG-/- cells are more sensitive to pemetrexed mediated intra S-phase arrest, DNA double strand break formation and apoptosis compared to UNG+/+ cells. Using data from western blots in chromatin extracts, PCNA staining of cells in S-phase, and pulse-chase labeling of replicating cells with CldU and IdU, we surmise that the accumulation of uracil in pemetrexed-treated UNG-/- cells is associated with significant replication fork instability. In addition, UNG-/- cells have reduced capacity to recover from pemetrexed-mediated DNA damage, as indicated by the persistence of S-phase arrest and gamma-H2AX foci. This defect in recovery was not explained by double strand break repair capacity, which was equivalent in UNG+/+ and UNG-/- cells. Using γ-H2AX ChIP sequencing, we observed a 5-fold increase in the number of γ-H2AX binding sites in UNG-/- cells compared to UNG+/+ cells treated at IC50 levels of pemetrexed. This analysis evinced distinct patterns of γ-H2AX binding in UNG+/+ and UNG-/- cells. Double strand breaks (γ-H2AX) were more significantly associated with transcription start sites and putative origins of replication in UNG-/- cells compared to UNG+/+ cells. Based on these data we conclude that uracil accumulation, and thus UNG activity, during pemetrexed exposure directs both the quantity and the location of double strand breaks. These findings support uracil mediated S-phase arrest and DNA replication fork collapse as the mechanism of double strand break formation and cell death in pemetrexed treated UNG-/- cells. Citation Format: Lachelle D. Weeks, Gabriel Zentner, Peter Scacheri, Stanton L. Gerson. Pemetrexed treatment results in DNA replication fork instability and double strand breaks formation in UNG-/- human cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4482. doi:10.1158/1538-7445.AM2013-4482

Meiotic recombination is initiated by the formation of numerous DNA double-strand breaks (DSBs) catalysed by the widely conserved Spo11 protein. In Saccharomyces cerevisiae, Spo11 requires nine other proteins for meiotic DSB formation; however, unlike Spo11, few of these are conserved across kingdoms. In order to investigate this recombination step in higher eukaryotes, we took advantage of a high-throughput meiotic mutant screen carried out in the model plant Arabidopsis thaliana. A collection of 55,000 mutant lines was screened, and spo11-like mutations, characterised by a drastic decrease in chiasma formation at metaphase I associated with an absence of synapsis at prophase, were selected. This screen led to the identification of two populations of mutants classified according to their recombination defects: mutants that repair meiotic DSBs using the sister chromatid such as Atdmc1 or mutants that are unable to make DSBs like Atspo11-1. We found that in Arabidopsis thaliana at least four proteins are necessary for driving meiotic DSB repair via the homologous chromosomes. These include the previously characterised DMC1 and the Hop1-related ASY1 proteins, but also the meiotic specific cyclin SDS as well as the Hop2 Arabidopsis homologue AHP2. Analysing the mutants defective in DSB formation, we identified the previously characterised AtSPO11-1, AtSPO11-2, and AtPRD1 as well as two new genes, AtPRD2 and AtPRD3. Our data thus increase the number of proteins necessary for DSB formation in Arabidopsis thaliana to five. Unlike SPO11 and (to a minor extent) PRD1, these two new proteins are poorly conserved among species, suggesting that the DSB formation mechanism, but not its regulation, is conserved among eukaryotes.

During meiosis, one round of DNA replication is followed by two rounds of division to create the gametes that are obligatory for biparental reproduction. To achieve the reductional segregation that is required during the first meiotic division, most eukaryotes utilise a program that involves the deliberate formation of DNA double‐stranded breaks in their genomes, followed by regulated homologous recombination and reciprocal exchange of genetic material between homologous chromosomes. This reciprocal exchange of genetic information physically links the homologous chromosomes to one another and provides the tension that is necessary for their segregation. Meiotic recombination is highly regulated to ensure that at least one reciprocal crossover event occurs between each pair of homologous chromosomes. Key Concepts Meiotic recombination physically links the homologous chromosomes to one another, creating the tension that is required for their segregation. Meiosis I is a reductional segregation that halves the ploidy of the cell. This process requires bi‐orientation of the homologous chromosomes but mono‐orientation of the sister chromatids, a feat that is achieved through structural modification of the sister chromatid centromeres by monopolin. Directed homologous recombination physically links the homologous chromosomes to one another, through preferential use of the homologous chromosome as the repair template, rather than the sister chromatid, followed by resolution of the recombination intermediate into a crossover. DNA double‐stranded break fate is decided early in the meiotic recombination pathway, with crossovers exclusively arising through a double‐Holliday junction intermediate and non‐crossovers primarily being formed through the synthesis‐dependent strand annealing pathway. Formation of the synaptonemal complex, a proteinaceous structure that assembles between homologous chromosomes during meiosis, is interdependent with meiotic recombination.

Meiosis is an essential process for sexually reproducing organisms, leading to the formation of specialized generative cells. This review intends to highlight current knowledge of early events during meiosis derived from various model organisms, including plants. It will particularly focus on cis- and trans-requirements of meiotic DNA double strand break (DSB) formation, a hallmark event during meiosis and a prerequisite for recombination of genetic traits. Proteins involved in DSB formation in different organisms, emphasizing the known factors from plants, will be introduced and their functions outlined. Recent technical advances in DSB detection and meiotic recombination analysis will be reviewed, as these new tools now allow analysis of early meiotic recombination in plants with incredible accuracy. To anticipate future directions in plant meiosis research, unpublished results will be included wherever possible.

Introduction: Targeted alpha therapy has shown promising results in preclinical and clinical studies. Alpha particle irradiation gives a high fraction of DNA double-strand breaks (DSB), as shown in vitro, resulting in a high probability of cell death. We have previously examined the therapeutic effects of 211At on solid colon carcinoma tumors (diameter approximately 1 cm), with tolerable activities (5 MBq/animals) resulting in non-palpable tumors within one week p.i. The aim of the present study was to investigate the formation of DNA DSB during tumor regression after radioimmunotherapy with 211At-mAb in a syngeneic rat colon carcinoma model. Methods: 18 rats bearing solid colon tumors (1 cm in diameter) between peritoneum and the abdominal muscle were injected intravenously with 5 MBq/animal 211At-BR96. Tumors were excised and paraffin-embedded after 10 min, 2 h, 8 h, 18 h, 24 h, and 48 h p.i. (3 tumors per time point). 53BP1 was stained by immunohistochemistry and used as a marker for DNA DSB. Untreated tumors were used as controls (n=9). DNA DSB were counted in central and peripheral tumor areas selected at random. Results: A few DNA DSB were detected in untreated tumors. Already 10 min p.i., the number of DNA DSB had increased slightly in peripheral tumor tissue. The number peaked 8 h p.i., when the number of DNA DSB had increased 50 times in the tumor periphery and 24 times in the tumor center. The number of DNA DSB then declined, but the difference between center and periphery remained, as expected considering the intratumoral distribution of radioimmunoconjugate. This correlates with the 211At half-life of 7.2 h. Conclusion: DNA DSB are formed early after injection of 211At-mAb and follows the intratumoral distribution of mAbs. Citation Format: Sophie E. Eriksson, Erika Elgström, Sture Lindegren, Tom Bäck. Formation of DNA double-strand breaks in colon tumors after targeted alpha therapy with 211At-mAb [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 834. doi:10.1158/1538-7445.AM2017-834

The Saccharomyces cerevisiae Dmc1 and Tid1 proteins are required for the pairing of homologous chromosomes during meiotic recombination. This pairing is the precursor to the formation of crossovers between homologs, an event that is necessary for the accurate segregation of chromosomes. Failure to form crossovers can have serious consequences and may lead to chromosomal imbalance. Dmc1, a meiosis-specific paralog of Rad51, mediates the pairing of homologous chromosomes. Tid1, a Rad54 paralog, although not meiosis-specific, interacts with Dmc1 and promotes crossover formation between homologs. In this study, we show that purified Dmc1 and Tid1 interact physically and functionally. Dmc1 forms stable nucleoprotein filaments that can mediate DNA strand invasion. Tid1 stimulates Dmc1-mediated formation of joint molecules. Under conditions optimal for Dmc1 reactions, Rad51 is specifically stimulated by Rad54, establishing that Dmc1-Tid1 and Rad51-Rad54 function as specific pairs. Physical interaction studies show that specificity in function is not dictated by direct interactions between the proteins. Our data are consistent with the hypothesis that Rad51-Rad54 function together to promote intersister DNA strand exchange, whereas Dmc1-Tid1 tilt the bias toward interhomolog DNA strand exchange.

DNA repair is known as a defense mechanism against genotoxic insults. However, the most lethal type of DNA damages, double-strand DNA breaks (DSBs), can be produced by DNA repair. We have previously demonstrated that when long patch base excision repair attempts to repair a synthetic substrate containing two uracils, the repair produces DSBs (Vispe, S. and Satoh, M. S. (2000) J. Biol. Chem. 275, 27386-27392 and Vispe, S., Ho, E. L., Yung, T. M., and Satoh, M. S. (2003) J. Biol. Chem. 278, 35279-35285). In this synthetic substrate, the two uracils are located on the opposite DNA strands (separated by an intervening sequence stable at 37 degrees C) and represent a high risk site for DSB formation. It is not clear, however, whether similar high risk sites are also induced in genomic DNA by exposure to DNA damaging agents. Thus, to investigate the mechanisms of DSB formation, we have modified the DSB formation assay developed previously and demonstrated that high risk sites for DSB formation are indeed generated in genomic DNA by exposure of cells to alkylating agents. In fact, genomic DNA containing alkylated base damages, which could represent high risk sites, are converted into DSBs by enzymes present in extracts prepared from cells derived from clinically normal individuals. Furthermore, DSBs are also produced by extracts from cells derived from ataxia-telangiectasia patients who show cancer proneness due to an impaired response to DSBs. These results suggest the presence of a novel link between base damage formation and DSBs and between long patch base excision repair and human diseases that occur due to an impaired response to DSB.

Meiotic recombination plays an essential role in the proper segregation of chromosomes at meiosis I in many sexually reproducing organisms. Meiotic recombination is initiated by the scheduled formation of genome-wide DNA double-strand breaks (DSBs). The timing of DSB formation is strictly controlled because unscheduled DSB formation is detrimental to genome integrity. Here, we investigated the role of DNA damage checkpoint mechanisms in the control of meiotic DSB formation using budding yeast. By using recombination defective mutants in which meiotic DSBs are not repaired, the effect of DNA damage checkpoint mutations on DSB formation was evaluated. The Tel1 (ATM) pathway mainly responds to unresected DSB ends, thus the sae2 mutant background in which DSB ends remain intact was employed. On the other hand, the Mec1 (ATR) pathway is primarily used when DSB ends are resected, thus the rad51 dmc1 double mutant background was employed in which highly resected DSBs accumulate. In order to separate the effect caused by unscheduled cell cycle progression, which is often associated with DNA damage checkpoint defects, we also employed the ndt80 mutation which permanently arrests the meiotic cell cycle at prophase I. In the absence of Tel1, DSB formation was reduced in larger chromosomes (IV, VII, II and XI) whereas no significant reduction was found in smaller chromosomes (III and VI). On the other hand, the absence of Rad17 (a critical component of the ATR pathway) lead to an increase in DSB formation (chromosomes VII and II were tested). We propose that, within prophase I, the Tel1 pathway facilitates DSB formation, especially in bigger chromosomes, while the Mec1 pathway negatively regulates DSB formation. We also identified prophase I exit, which is under the control of the DNA damage checkpoint machinery, to be a critical event associated with down-regulating meiotic DSB formation.

Meiotic recombination between homologous chromosomes in the yeast Saccharomyces cerevisiae is initiated by the formation of DNA double-strand breaks (DSBs). The mechanism of DSB formation and the factors that determine their frequency and location have yet to be elucidated. Current studies of meiotic recombination are also concerned with the question of the functional relationship between DSB formation and the other meiotic processes of homology searching, pairing and synapsis of homologues. To test if DNA identity is required for high levels of DSBs and recombination, we have asked whether small DNA heterologies (140-547 bp) located within the well characterized ARG4 initiator of meiotic recombination, can affect DSB formation and gene conversion events in the ARG4 locus. The present physical and genetic analyses show that some heterologies reduced recombination frequencies without altering DSB formation, whereas others reduced both DSB and gene conversion frequencies. These results suggest that DNA heterologies overlapping a recombination initiator impair meiotic gene conversion at two levels. First, some heterologies affect the level of DSB formation, revealing the existence of an anti-initiation process sensing the presence of sequence non-homology between the homologous chromosomes. Second, heterologies can impair the successful processing of the recombination intermediates once DSBs are made. We present a model for interhomologue cross-talks involving chromosomal and DNA/DNA interactions.

Purpose To investigate the relationship between abdominopelvic magnetic resonance (MR) imaging and formation of DNA double-strand breaks (DSBs) in peripheral blood lymphocytes among a cohort of healthy volunteers. Materials and Methods Blood samples were obtained from 40 healthy volunteers (23 women and 17 men; mean age, 27.2 years [range, 21-37 years]) directly before and 5 and 30 minutes after abdominopelvic MR imaging performed at 1.5 T (n = 20) or 3.0 T (n = 20). The number of DNA DSBs in isolated blood lymphocytes was quantified after indirect immunofluorescent staining of a generally accepted DSB marker, γ-H2AX, by means of high-throughput automated microscopy. As a positive control of DSB induction, blood lymphocytes from six volunteers were irradiated in vitro with x-rays at a dose of 1 Gy (70-90 keV). Statistical analysis was performed by using a Friedman test. Results No significant alteration in the frequency of DNA DSB induction was observed after MR imaging (before imaging: 0.22 foci per cell, interquartile range [IQR] = 0.54 foci per cell; 5 minutes after MR imaging: 0.08 foci per cell, IQR = 0.39 foci per cell; 30 minutes after MR imaging: 0.09 foci per cell, IQR = 0.63 foci per cell; P = .057). In vitro radiation of lymphocytes with 1 Gy led to a significant increase in DSBs (0.22 vs 3.43 foci per cell; P = .0312). The frequency of DSBs did not differ between imaging at 1.5 T and at 3.0 T (5 minutes after MR imaging: 0.23 vs 0.06 foci per cell, respectively [P = .57]; 30 minutes after MR imaging: 0.12 vs 0.08 foci per cell [P = .76]). Conclusion Abdominopelvic MR imaging performed at 1.5 T or 3.0 T does not affect the formation of DNA DSBs in peripheral blood lymphocytes.

DNA double-strand breaks (DSBs) can be lethal to a cell. However, most sexually reproducing organisms deliberately induce a substantial amount of developmentally programmed DSBs that are subsequently repaired via homologous recombination in meiosis. The goal of this self-damage and self-repair process is to establish physical connections between homologous chromosomes, thereby ensuring accurate chromosome segregation and producing haploid germ cells (sperm and eggs) (Fig. 1). Recombination also disrupts the linkage of polymorphisms on the same chromosome and thus promotes genome diversity and evolution. Alterations in normal recombination patterns cause human aneuploidy, and these errors are a major cause of spontaneous abortion and congenital birth defects.1 Figure 1. Overview of the events of meiosis. In meiosis I, homologous chromosomes exchange genetic information via recombination and are then segregated. In meiosis II, sister chromatids separate. Meiotic recombination is initiated by DNA double-strand ... Meiotic recombination does not occur randomly. It is more likely to happen in some genomic regions than others, largely due to nonrandom DSB distribution. There are large DSB-hot and -cold domains (tens of kilobases [kb]), within which are short regions called hotspots (typically several hundred base pairs wide), where DSBs preferentially form. This DSB landscape is shaped by a hierarchical combination of many factors including whole chromosome variation, large subchromosomal domains, cohesins and other chromosome structure proteins, chromatin structure, and local nucleotide composition.2 Notably, these factors act at different size scales and many of the molecular mechanisms connecting them to DSB formation are still not well understood. As potentially hazardous events, meiotic DSBs are tightly controlled in their timing, amount, and location. Emerging evidence in several organisms implies that the first step in recombination (DSB formation) is regulated by subsequent steps such as DSB repair. Recently, we explored such a feedback circuit in which homolog engagement shapes meiotic DSB number and spatial patterning in Saccharomyces cerevisiae (Fig. 1).3 For this purpose, we loosely define homolog engagement such that it could include the progression of recombination intermediates and/or the formation of synaptonemal complex (SC). (The SC is a meiosis-specific structure comprising the proteinaceous axes of a pair of homologous chromosomes held together by transverse filaments; it serves as a scaffold stabilizing the juxtaposition of homologous chromosomes and promotes the completion of recombination). Evidence in mice, flies and worms has suggested that negative feedback induced by engaging homologous chromosomes controls DSB formation.4-6 A test of this hypothesis arose from studies of the ZMM group of proteins (Zip1–4, Msh4–5, Mer3, Spo16, and Pph3). Mutants lacking ZMM proteins display defects in SC and recombination.7 We found that ZMM mutants formed a substantially greater number of DSBs, as judged by a variety of molecular and genetic assays. This was a surprise because ZMM proteins have traditionally been viewed as acting strictly downstream of DSB formation, but the new findings show that ZMMs are genetically both upstream and downstream. A simple way to explain these results is to propose that chromosomes stop making DSBs once they successfully engage their homologous partners. The homolog engagement defects in ZMM mutants allow chromosomes to continue making DSBs when normally they would have stopped. A plausible mechanism is that formation of SC leads to structural changes in chromosomes that inactivate or remove the DSB-forming machinery. Importantly, mapping of DSBs in zip3 mutants demonstrated that this feedback loop helps shape the genome-wide DSB distribution. Interestingly, different chromosomal subdomains responded differently to the DSB increase in zip3 mutants, with domains of greater or lesser change alternating along chromosomes. One possible explanation is that defective homolog engagement in zip3 mutants relieves the DSB suppression that would normally occur near sites of recombination. If so, this further implies that ZMM-dependent DSB suppression in wild-type cells spreads along chromosomes from sites of homolog engagement, with the magnitude of suppression decreasing with the distance from the engagement site. Besides homolog engagement, there are other regulatory elements shaping DSB distributions on different size scales.2 For example, few DSBs form in ~20-kb zones from telomeres, centromeres and the rDNA in wild-type. Despite a 1.8-fold increase of total DSBs in zip3 mutants, DSB frequencies in subtelomeric, and pericentromeric regions were elevated less than genome average, and were unchanged or reduced near the rDNA.3 Accordingly, ZMM-dependent DSB suppression is different from and subordinate to the DSB suppression mechanisms acting in these subdomains. Since DSB feedback circuits have been reported in other species as well, it will be interesting to map DSBs in mutants with feedback defects in other organisms, continuing the journey of exploring previously unknown regulators of the recombination landscape.

DNA is protected by packaging it into higher order chromatin fibres, but this can impede nuclear processes like DNA repair. Despite considerable research into the factors required for signalling and repairing DNA damage, it is unclear if there are concomitant changes in global chromatin fibre structure. In human cells DNA double strand break (DSB) formation triggers a signalling cascade resulting in H2AX phosphorylation (γH2AX), the rapid recruitment of chromatin associated proteins and the subsequent repair of damaged sites. KAP1 is a transcriptional corepressor and in HCT116 cells we found that after DSB formation by chemicals or ionising radiation there was a wave of, predominantly ATM dependent, KAP1 phosphorylation. Both KAP1 and phosphorylated KAP1 were readily extracted from cells indicating they do not have a structural role and γH2AX was extracted in soluble chromatin indicating that sites of damage are not attached to an underlying structural matrix. After DSB formation we did not find a concomitant change in the sensitivity of chromatin fibres to micrococcal nuclease digestion. Therefore to directly investigate higher order chromatin fibre structures we used a biophysical sedimentation technique based on sucrose gradient centrifugation to compare the conformation of chromatin fibres isolated from cells before and after DNA DSB formation. After damage we found global chromatin fibre compaction, accompanied by rapid linker histone dephosphorylation, consistent with fibres being more regularly folded or fibre deformation being stabilized by linker histones. We suggest that following DSB formation, although there is localised chromatin unfolding to facilitate repair, the bulk genome becomes rapidly compacted protecting cells from further damage.

Meiosis is the cell division programutilized by most sexually reproducingorganisms as a strategy to produce haploidgametes (i.e., sperm and eggs) from diploidparental cells. As suggested by its name,which stems from the Greek word mean-ing ‘‘to diminish or reduce’’, meiosisreduces the chromosome number by half.This is accomplished by following a singleround of DNA replication with twoconsecutive rounds of cell division (meiosisI and meiosis II). At meiosis I, homologouschromosomes segregate away from eachother (reductional division) and at meiosisII, sister chromatids segregate to oppositepoles of the spindle (equational division).During prophase of meiosis I, chromo-somes undergo a series of unique and well-orchestrated steps that promote accuratesegregation. These steps include the for-mation of programmed DNA double-strand breaks (DSBs), homologous chro-mosome pairing, and synapsis. A subset ofDSBs are repaired via recombinationbetween homologous chromosomes suchthat there is a reciprocal exchange ofgenetic material between the homologsresulting in crossover formation. Thesecrossover events, underpinned by flankingsister chromatid cohesion, generate phys-ical attachments between the homologs(chiasmata), which are important for theirproper alignment at the metaphase I plate.Either impaired DSB formation or afailure to form chiasmata during meiosiscan result in the formation of eggs andsperm carrying an incorrect number ofchromosomes, which in turn accounts fora large percentage of the miscarriages,birth defects, and infertility observed inhumans [1]. Thus, DSB formation is anessential process for successful offspringproduction. Although it is known thatDSB formation is catalyzed by Spo11, aconserved type II topoisomerase-like pro-tein [2,3], the regulation of DSB formationis not completely understood. In this issueof PLoS Genetics, Lake et al. [4] shed newlight on this process by identifying TradeEmbargo (Trem) as a critical protein forDSB production during Drosophila femalemeiosis.Recent studies in yeast have started touncover the molecular basis for the regu-lation of DSB induction. It is known that atleast ten proteins (Spo11-Ski8, Mer2-Mei4-Rec114, Rec102-Rec104, Mre11-Rad50-Xrs2) are essential for DSB induction inSaccharomyces cerevisiae [5]. S phase cyclin-dependent kinase Cdc28–Clb5/Clb6(CDK-S) and the Dbf4-dependent kinaseCdc7–Dbf4 (DDK) regulate the timing ofDSB formation [6,7]. Mer2 is an essentialtarget of both CDK-S and DDK. Specif-ically, Mer2 is phosphorylated by CDK-Sat Ser30. This phosphorylation primesMer2 for subsequent phosphorylation byDDK on Ser29, creating a negativelycharged ‘‘patch’’. This coordinated phos-phorylationtriggerstheinteractionofMer2with Mei4 and Rec114 [6,7]. CDK-S-mediated phosphorylation of Mer2 is alsoimportant for promoting the interactionbetween Mer2 and Xrs2 [8].Thus, pS30ofMer2 recruits the Mre11-Rad50-Xrs2complex to DSB hotspots. Finally, Spo11-Ski8 and Rec102-Rec104 sub-complexesare recruited to the hotspots.Efforts to identify DSB-inducing factorsin other species have been hampered inpart by the low level of sequence conser-vation shared with the factors first identi-fied in S. cerevisiae. However, a sophisticat-ed in silico analysis recently identified theorthologs of Mei4 and Rec114 in fissionyeast, plants, and mammals [9]. Similar toyeast, mei42/2 mice lack meiotic DSBinduction [9]. In mammals, it has beenreported that Prdm9/Meisetz, which is amulti zinc-finger protein that containsKRAB and methyl transferase domains,marks DSB hotspots [10–12]. Moreover,the polymorphism of the zinc fingers altersthe binding activity to hotspot sequences[10,11,12], although Prdm9 is not essen-tial for DSB formation [13]. In othermodel organisms, HIM-17 which is a sixTHAP (C2CH) repeat containing proteinin Caenorhabditis elegans [14], MEI1 [15] inmice, and SWI1 in Arabidopsis thaliana [16]have been reported as factors required forDSB formation. However, how theseproteins act to make DSBs remainsunclear.