Modeling homologous chromosome recognition via nonspecific interactions.

In many organisms, most notably Drosophila, homologous chromosomes associate in somatic cells, a phenomenon known as somatic pairing, which takes place without double strand breaks or strand invasion, thus requiring some other mechanism for homologs to recognize each other. Several studies have suggested a "specific button" model, in which a series of distinct regions in the genome, known as buttons, can associate with each other, mediated by different proteins that bind to these different regions. Here, we use computational modeling to evaluate an alternative "button barcode" model, in which there is only one type of recognition site or adhesion button, present in many copies in the genome, each of which can associate with any of the others with equal affinity. In this model, buttons are nonuniformly distributed, such that alignment of a chromosome with its correct homolog, compared with a nonhomolog, is energetically favored; since to achieve nonhomologous alignment, chromosomes would be required to mechanically deform in order to bring their buttons into mutual register. By simulating randomly generated nonuniform button distributions, many highly effective button barcodes can be easily found, some of which achieve virtually perfect pairing fidelity. This model is consistent with existing literature on the effect of translocations of different sizes on homolog pairing. We conclude that a button barcode model can attain highly specific homolog recognition, comparable to that seen in actual cells undergoing somatic homolog pairing, without the need for specific interactions. This model may have implications for how meiotic pairing is achieved.

Screening and characterization of 133 physiologically-relevant environmental chemicals for reproductive toxicity.

Reproduction is a functional outcome that relies on complex cellular, tissue, and organ interactions that span the developmental period to adulthood. Thus, the assessment of its disruption by environmental chemicals would benefit significantly from scalable and innovative approaches to testing using functionally comparable reproductive models such as the nematode C. elegans. We adapted a previously described low-throughput in vivo chromosome segregation assay using C. elegans predictive of reproductive toxicity and leveraged available public data sources (ToxCast, ICE) to screen and characterize 133 physiologically-relevant chemicals in a high-throughput manner. The screening outcome was further validated in a second, independent in vivo assay assessing embryonic viability. In total, 13 chemicals were classified as reproductive toxicants with the two most active chemicals belonging to the large family of Quaternary Ammonium Compounds (QACs) commonly used as disinfectants but with limited available reproductive toxicity data. We compared the results from the C. elegans assay with ToxCast in vitro data compiled from 700+ cell response assays and 300+ signaling pathways-based assays. We did not observe a difference in the bioactivity or in the average potency (AC50) between the top and bottom chemicals. However, the intended target categories were significantly different between the classified chemicals with, in particular, an over-representation of steroid hormone targets for the high Z-score chemicals. Taken together, these results point to the value of in vivo models that scale to high-throughput level for reproductive toxicity assessment and to the need to prioritize the assessment of QACs impacts on reproduction.

A high-throughput method for quantifying Drosophila fecundity.

Measurements of Drosophila fecundity are used in a wide variety of studies, such as investigations of stem cell biology, nutrition, behavior, and toxicology. In addition, because fecundity assays are performed on live flies, they are suitable for longitudinal studies such as investigations of aging or prolonged chemical exposure. However, standard Drosophila fecundity assays have been difficult to perform in a high-throughput manner because experimental factors such as the physiological state of the flies and environmental cues must be carefully controlled to achieve consistent results. In addition, exposing flies to a large number of different experimental conditions (such as chemical additives in the diet) and manually counting the number of eggs laid to determine the impact on fecundity is time-consuming. We have overcome these challenges by combining a new multiwell fly culture strategy with a novel 3D-printed fly transfer device to rapidly and accurately transfer flies from one plate to another; the RoboCam, a low-cost, custom built robotic camera to capture images of the wells automatically; and an image segmentation pipeline to automatically identify and quantify eggs. We show that this method is compatible with robust and consistent egg laying throughout the assay period; and demonstrate that the automated pipeline for quantifying fecundity is very accurate (r2 = 0.98 for the correlation between the automated egg counts and the ground truth) In addition, we show that this method can be used to efficiently detect the effects on fecundity induced by dietary exposure to chemicals. Taken together, this strategy substantially increases the efficiency and reproducibility of high throughput egg laying assays that require exposing flies to multiple different media conditions.

Screening and characterization of 133 physiologically-relevant environmental chemicals for reproductive toxicity.

Reproduction is a functional outcome that relies on complex cellular, tissue, and organ interactions that span the developmental period to adulthood. Thus, the assessment of its disruption by environmental chemicals is remarkably painstaking in conventional toxicological animal models and does not scale up to the number of chemicals present in our environment and requiring testing. We adapted a previously described low-throughput in vivo chromosome segregation assay using C. elegans predictive of reproductive toxicity and leveraged available public data sources (ToxCast, ICE) to screen and characterize 133 physiologically-relevant chemicals in a high-throughput manner. The screening outcome was further validated in a second, independent in vivo assay assessing embryonic viability. In total, 13 chemicals were classified as reproductive toxicants with the two most active chemicals belonging to the large family of Quaternary Ammonium Compounds (QACs) commonly used as disinfectants but with limited available reproductive toxicity data. We compared the results from the C. elegans assay with ToxCast in vitro data compiled from 700+ cell response assays and 300+ signaling pathways-based assays. We did not observe a difference in the bioactivity or in average potency (AC50) between the top and bottom chemicals. However, the intended target categories were significantly different between the classified chemicals with, in particular, an over-representation of steroid hormone targets for the high Z-score chemicals. Taken together, these results point to the value of in vivo models that scale to high-throughput level for reproductive toxicity assessment and to the need to prioritize the assessment of QACs impacts on reproduction.

Homologous chromosome recognition via nonspecific interactions.

In many organisms, most notably Drosophila, homologous chromosomes in somatic cells associate with each other, a phenomenon known as somatic homolog pairing. Unlike in meiosis, where homology is read out at the level of DNA sequence complementarity, somatic homolog pairing takes place without double strand breaks or strand invasion, thus requiring some other mechanism for homologs to recognize each other. Several studies have suggested a "specific button" model, in which a series of distinct regions in the genome, known as buttons, can associate with each other, presumably mediated by different proteins that bind to these different regions. Here we consider an alternative model, which we term the "button barcode" model, in which there is only one type of recognition site or adhesion button, present in many copies in the genome, each of which can associate with any of the others with equal affinity. An important component of this model is that the buttons are non-uniformly distributed, such that alignment of a chromosome with its correct homolog, compared with a non-homolog, is energetically favored; since to achieve nonhomologous alignment, chromosomes would be required to mechanically deform in order to bring their buttons into mutual register. We investigated several types of barcodes and examined their effect on pairing fidelity. We found that high fidelity homolog recognition can be achieved by arranging chromosome pairing buttons according to an actual industrial barcode used for warehouse sorting. By simulating randomly generated non-uniform button distributions, many highly effective button barcodes can be easily found, some of which achieve virtually perfect pairing fidelity. This model is consistent with existing literature on the effect of translocations of different sizes on homolog pairing. We conclude that a button barcode model can attain highly specific homolog recognition, comparable to that seen in actual cells undergoing somatic homolog pairing, without the need for specific interactions. This model may have implications for how meiotic pairing is achieved.

Kinetic analysis of synaptonemal complex dynamics during meiosis of yeast Saccharomyces cerevisiae reveals biphasic growth and abortive disassembly.

The synaptonemal complex (SC) is a dynamic structure formed between chromosomes during meiosis which stabilizes and supports many essential meiotic processes such as pairing and recombination. In budding yeast, Zip1 is a functionally conserved element of the SC that is important for synapsis. Here, we directly measure the kinetics of Zip1-GFP assembly and disassembly in live cells of the yeast S. cerevisiae. The imaging of SC assembly in yeast is challenging due to the large number of chromosomes packed into a small nucleus. We employ a zip3Δ mutant in which only a few chromosomes undergo synapsis at any given time, initiating from a single site on each chromosome, thus allowing the assembly and disassembly kinetics of single SCs to be accurately monitored in living cells. SC assembly occurs with both monophasic and biphasic kinetics, in contrast to the strictly monophasic assembly seen in C. elegans. In wild-type cells, once maximal synapsis is achieved, programmed final disassembly rapidly follows, as Zip1 protein is actively degraded. In zip3Δ, this period is extended and final disassembly is prolonged. Besides final disassembly, we found novel disassembly events involving mostly short SCs that disappeared in advance of programmed final disassembly, which we termed "abortive disassembly." Abortive disassembly is distinct from final disassembly in that it occurs when Zip1 protein levels are still high, and exhibits a much slower rate of disassembly, suggesting a different mechanism for removal in the two types of disassembly. We speculate that abortive disassembly events represent defective or stalled SCs, possibly representing SC formation between non-homologs, that is then targeted for dissolution. These results reveal novel aspects of SC assembly and disassembly, potentially providing evidence of additional regulatory pathways controlling not just the assembly, but also the disassembly, of this complex cellular structure.

Modeling cell biological features of meiotic chromosome pairing to study interlock resolution.

During meiosis, homologous chromosomes become associated side by side in a process known as homologous chromosome pairing. Pairing requires long range chromosome motion through a nucleus that is full of other chromosomes. It remains unclear how the cell manages to align each pair of chromosomes quickly while mitigating and resolving interlocks. Here, we use a coarse-grained molecular dynamics model to investigate how specific features of meiosis, including motor-driven telomere motion, nuclear envelope interactions, and increased nuclear size, affect the rate of pairing and the mitigation/resolution of interlocks. By creating in silico versions of three yeast strains and comparing the results of our model to experimental data, we find that a more distributed placement of pairing sites along the chromosome is necessary to replicate experimental findings. Active motion of the telomeric ends speeds up pairing only if binding sites are spread along the chromosome length. Adding a meiotic bouquet significantly speeds up pairing but does not significantly change the number of interlocks. An increase in nuclear size slows down pairing while greatly reducing the number of interlocks. Interestingly, active forces increase the number of interlocks, which raises the question: How do these interlocks resolve? Our model gives us detailed movies of interlock resolution events which we then analyze to build a step-by-step recipe for interlock resolution. In our model, interlocks must first translocate to the ends, where they are held in a quasi-stable state by a large number of paired sites on one side. To completely resolve an interlock, the telomeres of the involved chromosomes must come in close proximity so that the cooperativity of pairing coupled with random motion causes the telomeres to unwind. Together our results indicate that computational modeling of homolog pairing provides insight into the specific cell biological changes that occur during meiosis.

Steroid hormones regulate genome-wide epigenetic programming and gene transcription in human endometrial cells with marked aberrancies in endometriosis.

Programmed cellular responses to cycling ovarian-derived steroid hormones are central to normal endometrial function. Abnormalities therein, as in the estrogen-dependent, progesterone-"resistant" disorder, endometriosis, predispose to infertility and poor pregnancy outcomes. The endometrial stromal fibroblast (eSF) is a master regulator of pregnancy success. However, the complex hormone-epigenome-transcriptome interplay in eSF by each individual steroid hormone, estradiol (E2) and/or progesterone (P4), under physiologic and pathophysiologic conditions, is poorly understood and was investigated herein. Genome-wide analysis in normal, early and late stage eutopic eSF revealed: i) In contrast to P4, E2 extensively affected the eSF DNA methylome and transcriptome. Importantly, E2 resulted in a more open versus closed chromatin, confirmed by histone modification analysis. Combined E2 with P4 affected a totally different landscape than E2 or P4 alone. ii) P4 responses were aberrant in early and late stage endometriosis, and mapping differentially methylated CpG sites with progesterone receptor targets from the literature revealed different but not decreased P4-targets, leading to question the P4-"resistant" phenotype in endometriosis. Interestingly, an aberrant E2-response was noted in eSF from endometriosis women; iii) Steroid hormones affected specific genomic contexts and locations, significantly enriching enhancers and intergenic regions and minimally involving proximal promoters and CpG islands, regardless of hormone type and eSF disease state. iv) In eSF from women with endometriosis, aberrant hormone-induced methylation signatures were mainly due to existing DNA methylation marks prior to hormone treatments and involved known endometriosis genes and pathways. v) Distinct DNA methylation and transcriptomic signatures revealed early and late stage endometriosis comprise unique disease subtypes. Taken together, the data herein, for the first time, provide significant insight into the hormone-epigenome-transcriptome interplay of each steroid hormone in normal eSF, and aberrant E2 response, distinct disease subtypes, and pre-existing epigenetic aberrancies in the setting of endometriosis, provide mechanistic insights into how endometriosis affects endometrial function/dysfunction.

Network Rewiring of Homologous Recombination Enzymes during Mitotic Proliferation and Meiosis.

Homologous recombination (HR) is essential for high-fidelity DNA repair during mitotic proliferation and meiosis. Yet, context-specific modifications must tailor the recombination machinery to avoid (mitosis) or enforce (meiosis) the formation of reciprocal exchanges-crossovers-between recombining chromosomes. To obtain molecular insight into how crossover control is achieved, we affinity purified 7 DNA-processing enzymes that channel HR intermediates into crossovers or noncrossovers from vegetative cells or cells undergoing meiosis. Using mass spectrometry, we provide a global characterization of their composition and reveal mitosis- and meiosis-specific modules in the interaction networks. Functional analyses of meiosis-specific interactors of MutLγ-Exo1 identified Rtk1, Caf120, and Chd1 as regulators of crossing-over. Chd1, which transiently associates with Exo1 at the prophase-to-metaphase I transition, enables the formation of MutLγ-dependent crossovers through its conserved ability to bind and displace nucleosomes. Thus, rewiring of the HR network, coupled to chromatin remodeling, promotes context-specific control of the recombination outcome.

Modeling meiotic chromosome pairing: a tug of war between telomere forces and a pairing-based Brownian ratchet leads to increased pairing fidelity.

Meiotic homolog pairing involves associations between homologous DNA regions scattered along the length of a chromosome. When homologs associate, they tend to do so by a processive zippering process, which apparently results from avidity effects. Using a computational model, we show that this avidity-driven processive zippering reduces the selectivity of pairing. When active random forces are applied to telomeres, this drop in selectivity is eliminated in a force-dependent manner. Further simulations suggest that active telomere forces are engaged in a tug-of-war against zippering, which can be interpreted as a Brownian ratchet with a stall force that depends on the dissociation constant of pairing. When perfectly homologous regions of high affinity compete with homeologous regions of lower affinity, the affinity difference can be amplified through this tug of war effect provided the telomere force acts in a range that is strong enough to oppose zippering of homeologs while still permitting zippering of correct homologs. The degree of unzippering depends on the radius of the nucleus, such that complete unzippering of homeologous regions can only take place if the nucleus is large enough to pull the two chromosomes completely apart. A picture of meiotic pairing thus emerges that is fundamentally mechanical in nature, possibly explaining the purpose of active telomere forces, increased nuclear diameter, and the presence of 'Maverick' chromosomes in meiosis.

Regulated Crossing-Over Requires Inactivation of Yen1/GEN1 Resolvase during Meiotic Prophase I.

During meiosis, crossover recombination promotes the establishment of physical connections between homologous chromosomes, enabling their bipolar segregation. To ensure that persistent recombination intermediates are disengaged prior to the completion of meiosis, the Yen1(GEN1) resolvase is strictly activated at the onset of anaphase II. Whether controlled activation of Yen1 is important for meiotic crossing-over is unknown. Here, we show that CDK-mediated phosphorylation of Yen1 averts its pervasive recruitment to recombination intermediates during prophase I. Yen1 mutants that are refractory to phosphorylation resolve DNA joint molecules prematurely and form crossovers independently of MutLγ, the central crossover resolvase during meiosis. Despite bypassing the requirement for MutLγ in joint molecule processing and promoting crossover-specific resolution, unrestrained Yen1 impairs the spatial distribution of crossover events, genome-wide. Thus, active suppression of Yen1 function, and by inference also of Mus81-Mms4(EME1) and Slx1-Slx4(BTBD12) resolvases, avoids precocious resolution of recombination intermediates to enable meiotic crossover patterning.

Kinetochores: Importance of Being Fashionably Late.

A model explains how a critical delay before microtubules detach from kinetochores during cell division might be achieved.

In Vivo Imaging of Budding Yeast Meiosis.

Tracking biological events in living cells provides kinetic information about biological processes that can be missed in more traditional methods using fixed samples at designated time intervals. Here we describe a methodology for in vivo fluorescence microscopy of yeast cells undergoing meiosis. This method allows tracking of individual cells over extended periods of time through every stage of the meiotic transformation while minimizing phototoxicity and sustaining conditions that support meiotic growth.

Modeling meiotic chromosome pairing: nuclear envelope attachment, telomere-led active random motion, and anomalous diffusion.

The recognition and pairing of homologous chromosomes during meiosis is a complex physical and molecular process involving a combination of polymer dynamics and molecular recognition events. Two highly conserved features of meiotic chromosome behavior are the attachment of telomeres to the nuclear envelope and the active random motion of telomeres driven by their interaction with cytoskeletal motor proteins. Both of these features have been proposed to facilitate the process of homolog pairing, but exactly what role these features play in meiosis remains poorly understood. Here we investigate the roles of active motion and nuclear envelope tethering using a Brownian dynamics simulation in which meiotic chromosomes are represented by a Rouse polymer model subjected to tethering and active forces at the telomeres. We find that tethering telomeres to the nuclear envelope slows down pairing relative to the rates achieved by unattached chromosomes, but that randomly directed active forces applied to the telomeres speed up pairing dramatically in a manner that depends on the statistical properties of the telomere force fluctuations. The increased rate of initial pairing cannot be explained by stretching out of the chromosome conformation but instead seems to correlate with anomalous diffusion of sub-telomeric regions.

DAZL and CPEB1 regulate mRNA translation synergistically during oocyte maturation.

Meiotic progression requires exquisitely coordinated translation of maternal messenger (m)RNA that has accumulated during oocyte growth. A major regulator of this program is the cytoplasmic polyadenylation element binding protein 1 (CPEB1). However, the temporal pattern of translation at different meiotic stages indicates the function of additional RNA binding proteins (RBPs). Here, we report that deleted in azoospermia-like (DAZL) cooperates with CPEB1 to regulate maternal mRNA translation. Using a strategy that monitors ribosome loading onto endogenous mRNAs and a prototypic translation target, we show that ribosome loading is induced in a DAZL- and CPEB1-dependent manner, as the oocyte reenters meiosis. Depletion of the two RBPs from oocytes and mutagenesis of the 3' untranslated regions (UTRs) demonstrate that both RBPs interact with the Tex19.1 3' UTR and cooperate in translation activation of this mRNA. We observed a synergism between DAZL and cytoplasmic polyadenylation elements (CPEs) in the translation pattern of maternal mRNAs when using a genome-wide analysis. Mechanistically, the number of DAZL proteins loaded onto the mRNA and the characteristics of the CPE might define the degree of cooperation between the two RBPs in activating translation and meiotic progression.

Reduced Crossover Interference and Increased ZMM-Independent Recombination in the Absence of Tel1/ATM.

Meiotic recombination involves the repair of double-strand break (DSB) precursors as crossovers (COs) or noncrossovers (NCOs). The proper number and distribution of COs is critical for successful chromosome segregation and formation of viable gametes. In budding yeast the majority of COs occurs through a pathway dependent on the ZMM proteins (Zip2-Zip3-Zip4-Spo16, Msh4-Msh5, Mer3), which form foci at CO-committed sites. Here we show that the DNA-damage-response kinase Tel1/ATM limits ZMM-independent recombination. By whole-genome mapping of recombination products, we find that lack of Tel1 results in higher recombination and reduced CO interference. Yet the number of Zip3 foci in tel1Δ cells is similar to wild type, and these foci show normal interference. Analysis of recombination in a tel1Δ zip3Δ double mutant indicates that COs are less dependent on Zip3 in the absence of Tel1. Together these results reveal that in the absence of Tel1, a significant proportion of COs occurs through a non-ZMM-dependent pathway, contributing to a CO landscape with poor interference. We also see a significant change in the distribution of all detectable recombination products in the absence of Tel1, Sgs1, Zip3, or Msh4, providing evidence for altered DSB distribution. These results support the previous finding that DSB interference depends on Tel1, and further suggest an additional level of DSB interference created through local repression of DSBs around CO-designated sites.

Follicle dynamics and global organization in the intact mouse ovary.

Quantitative analysis of tissues and organs can reveal large-scale patterning as well as the impact of perturbations and aging on biological architecture. Here we develop tools for imaging of single cells in intact organs and computational approaches to assess spatial relationships in 3D. In the mouse ovary, we use nuclear volume of the oocyte to read out quiescence or growth of oocyte-somatic cell units known as follicles. This in-ovary quantification of non-growing follicle dynamics from neonate to adult fits a mathematical function, which corroborates the model of fixed oocyte reserve. Mapping approaches show that radial organization of folliculogenesis established in the newborn ovary is preserved through adulthood. By contrast, inter-follicle clustering increases during aging with different dynamics depending on size. These broadly applicable tools can reveal high dimensional phenotypes and age-related architectural changes in other organs. In the adult mouse pancreas, we find stochastic radial organization of the islets of Langerhans but evidence for localized interactions among the smallest islets.

Integrity of the yeast mitochondrial genome, but not its distribution and inheritance, relies on mitochondrial fission and fusion.

Mitochondrial DNA (mtDNA) is essential for mitochondrial and cellular function. In Saccharomyces cerevisiae, mtDNA is organized in nucleoprotein structures termed nucleoids, which are distributed throughout the mitochondrial network and are faithfully inherited during the cell cycle. How the cell distributes and inherits mtDNA is incompletely understood although an involvement of mitochondrial fission and fusion has been suggested. We developed a LacO-LacI system to noninvasively image mtDNA dynamics in living cells. Using this system, we found that nucleoids are nonrandomly spaced within the mitochondrial network and observed the spatiotemporal events involved in mtDNA inheritance. Surprisingly, cells deficient in mitochondrial fusion and fission distributed and inherited mtDNA normally, pointing to alternative pathways involved in these processes. We identified such a mechanism, where we observed fission-independent, but F-actin-dependent, tip generation that was linked to the positioning of mtDNA to the newly generated tip. Although mitochondrial fusion and fission were dispensable for mtDNA distribution and inheritance, we show through a combination of genetics and next-generation sequencing that their absence leads to an accumulation of mitochondrial genomes harboring deleterious structural variations that cluster at the origins of mtDNA replication, thus revealing crucial roles for mitochondrial fusion and fission in maintaining the integrity of the mitochondrial genome.

Controlling meiotic recombinational repair - specifying the roles of ZMMs, Sgs1 and Mus81/Mms4 in crossover formation.

Crossovers (COs) play a critical role in ensuring proper alignment and segregation of homologous chromosomes during meiosis. How the cell balances recombination between CO vs. noncrossover (NCO) outcomes is not completely understood. Further lacking is what constrains the extent of DNA repair such that multiple events do not arise from a single double-strand break (DSB). Here, by interpreting signatures that result from recombination genome-wide, we find that synaptonemal complex proteins promote crossing over in distinct ways. Our results suggest that Zip3 (RNF212) promotes biased cutting of the double Holliday-junction (dHJ) intermediate whereas surprisingly Msh4 does not. Moreover, detailed examination of conversion tracts in sgs1 and mms4-md mutants reveal distinct aberrant recombination events involving multiple chromatid invasions. In sgs1 mutants, these multiple invasions are generally multichromatid involving 3-4 chromatids; in mms4-md mutants the multiple invasions preferentially resolve into one or two chromatids. Our analysis suggests that Mus81/Mms4 (Eme1), rather than just being a minor resolvase for COs is crucial for both COs and NCOs in preventing chromosome entanglements by removing 3'- flaps to promote second-end capture. Together our results force a reevaluation of how key recombination enzymes collaborate to specify the outcome of meiotic DNA repair.

Down-regulation of Rad51 activity during meiosis in yeast prevents competition with Dmc1 for repair of double-strand breaks.

Interhomolog recombination plays a critical role in promoting proper meiotic chromosome segregation but a mechanistic understanding of this process is far from complete. In vegetative cells, Rad51 is a highly conserved recombinase that exhibits a preference for repairing double strand breaks (DSBs) using sister chromatids, in contrast to the conserved, meiosis-specific recombinase, Dmc1, which preferentially repairs programmed DSBs using homologs. Despite the different preferences for repair templates, both Rad51 and Dmc1 are required for interhomolog recombination during meiosis. This paradox has recently been explained by the finding that Rad51 protein, but not its strand exchange activity, promotes Dmc1 function in budding yeast. Rad51 activity is inhibited in dmc1Δ mutants, where the failure to repair meiotic DSBs triggers the meiotic recombination checkpoint, resulting in prophase arrest. The question remains whether inhibition of Rad51 activity is important during wild-type meiosis, or whether inactivation of Rad51 occurs only as a result of the absence of DMC1 or checkpoint activation. This work shows that strains in which mechanisms that down-regulate Rad51 activity are removed exhibit reduced numbers of interhomolog crossovers and noncrossovers. A hypomorphic mutant, dmc1-T159A, makes less stable presynaptic filaments but is still able to mediate strand exchange and interact with accessory factors. Combining dmc1-T159A with up-regulated Rad51 activity reduces interhomolog recombination and spore viability, while increasing intersister joint molecule formation. These results support the idea that down-regulation of Rad51 activity is important during meiosis to prevent Rad51 from competing with Dmc1 for repair of meiotic DSBs.