Meiotic crossover patterns: Obligatory crossover,interference and homeostasis in a single process

During meiosis, crossover recombination is tightly regulated. A spatial patterning phenomenon known as interference ensures that crossovers are well-spaced along the chromosomes. Additionally, every pair of homologs acquires at least one crossover. A third feature, crossover homeostasis, buffers the system such that the number of crossovers remains steady despite decreases or increases in the number of earlier recombinational interactions. Here we summarize recent work from our laboratory supporting the idea that all 3 of these aspects are intrinsic consequences of a single basic process and suggesting that the underlying logic of this process corresponds to that embodied in a particular (beam-film) model.     

Heterozygous insertions alter crossover distribution but allow crossover interference in Caenorhabditis elegans

The normal distribution of crossover events on meiotic bivalents depends on homolog recognition, alignment, and interference. We developed a method for precisely locating all crossovers on Caenorhabditis elegans chromosomes and demonstrated that wild-type animals have essentially complete interference, with each bivalent receiving one and only one crossover. A physical break in one homolog has previously been shown to disrupt interference, suggesting that some aspect of bivalent structure is required for interference. We measured the distribution of crossovers in animals heterozygous for a large insertion to determine whether a break in sequence homology would have the same effect as a physical break. Insertions disrupt crossing over locally. However, every bivalent still experiences essentially one and only one crossover, suggesting that interference can act across a large gap in homology. Although insertions did not affect crossover number, they did have an effect on crossover distribution. Crossing over was consistently higher on the side of the chromosome bearing the homolog recognition region and lower on the other side of the chromosome. We suggest that nonhomologous sequences cause heterosynapsis, which disrupts crossovers along the distal chromosome, even when those regions contain sequences that could otherwise align. However, because crossovers are not completely eliminated distal to insertions, we propose that alignment can be reestablished after a megabase-scale gap in sequence homology.     

The flexibility of DNA double crossover molecules

Double crossover molecules are DNA structures containing two Holliday junctions connected by two double helical arms. There are several types of double crossover molecules, differentiated by the relative orientations of their helix axes, parallel or antiparallel, and by the number of double helical half-turns (even or odd) between the two crossovers. They are found as intermediates in meiosis and they have been used extensively in structural DNA nanotechnology for the construction of one-dimensional and two-dimensional arrays and in a DNA nanomechanical device. Whereas the parallel double helical molecules are usually not well behaved, we have focused on the antiparallel molecules; antiparallel molecules with an even number of half-turns between crossovers (termed DAE molecules) produce a reporter strand when ligated, facilitating their characterization in a ligation cyclization assay. Hence, we have estimated the flexibility of antiparallel DNA double crossover molecules by means of ligation-closure experiments. We are able to show that these molecules are approximately twice as rigid as linear duplex DNA.     

Two meiotic crossover classes cohabit in Arabidopsis: one is dependent on MER3,whereas the other one is not

BACKGROUND: Crossovers are essential for the completion of meiosis. Recently, two pathways of crossover formation have been identified on the basis of distinct genetic controls. In one pathway, crossover inhibits the occurrence of another such event in a distance-dependent manner. This phenomenon is known as interference. The second kind of crossover is insensitive to interference. The two pathways function independently in budding yeast. Only interference-insensitive crossovers occur in Schizosaccharomyces pombe. In contrast, only interference-sensitive crossovers occur in Caenorabditis elegans. The situation in mammals and plants remains unclear. Mer3 is one of the genes shown to be required for the formation of interference-sensitive crossovers in Saccharomyces cerevisiae. RESULTS: To unravel the crossover status in the plant Arabidopsis thaliana, we investigated the role of the A. thaliana MER3 gene through the characterization of a series of allelic mutants. All mer3 mutants showed low levels of fertility and a significant decrease (about 75%) but not a total disappearance of meiotic crossovers, with the number of recombination events initiated in the mutants being similar to that in the wild-type. Genetic analyses showed that the residual crossovers in mer3 mutants did not display interference in one set of adjacent intervals. CONCLUSIONS: Mutation in MER3 in Arabidopsis appeared to be specific to recombination events resulting in interference-sensitive crossovers. Thus, MER3 function is conserved from yeast to plants and may exist in other metazoans. Arabidopsis therefore has at least two pathways for crossover formation, one giving rise to interference-sensitive crossover and the other to independently distributed crossovers.     

Drosophila PCH2 is required for a pachytene checkpoint that monitors double-strand-break-independent events leading to meiotic crossover formation

During meiosis, programmed DNA double-strand breaks (DSBs) are repaired to create at least one crossover per chromosome arm. Crossovers mature into chiasmata, which hold and orient the homologous chromosomes on the meiotic spindle to ensure proper segregation at meiosis I. This process is usually monitored by one or more checkpoints that ensure that DSBs are repaired prior to the meiotic divisions. We show here that mutations in Drosophila genes required to process DSBs into crossovers delay two important steps in meiotic progression: a chromatin-remodeling process associated with DSB formation and the final steps of oocyte selection. Consistent with the hypothesis that a checkpoint has been activated, the delays in meiotic progression are suppressed by a mutation in the Drosophila homolog of pch2. The PCH2-dependent delays also require proteins thought to regulate the number and distribution of crossovers, suggesting that this checkpoint monitors events leading to crossover formation. Surprisingly, two lines of evidence suggest that the PCH2-dependent checkpoint does not reflect the accumulation of unprocessed recombination intermediates: the delays in meiotic progression do not depend on DSB formation or on mei-41, the Drosophila ATR homolog, which is required for the checkpoint response to unrepaired DSBs. We propose that the sites and/or conditions required to promote crossovers are established independently of DSB formation early in meiotic prophase. Furthermore, the PCH2-dependent checkpoint is activated by these events and pachytene progression is delayed until the DSB repair complexes required to generate crossovers are assembled. Interestingly, PCH2-dependent delays in prophase may allow additional crossovers to form.     

Negative crossover interference in maize translocation heterozygotes.

Negative interference describes a situation where two genetic regions have more double crossovers than would be expected considering the crossover rate of each region. We detected negative crossover interference while attempting to genetically map translocation breakpoints in maize. In an attempt to find precedent examples we determined there was negative interference among previously published translocation breakpoint mapping data in maize. It appears that negative interference was greater when the combined map length of the adjacent regions was smaller. Even positive interference appears to have been reduced when the combined lengths of adjacent regions were below 40 cM. Both phenomena can be explained by a reduction in crossovers near the breakpoints or, more specifically, by a failure of regions near breakpoints to become competent for crossovers. A mathematical explanation is provided.     

Global analysis of the meiotic crossover landscape

Tight control of the number and distribution of crossovers is of great importance for meiosis. Crossovers establish chiasmata, which are physical connections between homologous chromosomes that provide the tension necessary to align chromosomes on the meiotic spindle. Understanding the mechanisms underlying crossover control has been hampered by the difficulty in determining crossover distributions. Here, we present a microarray-based method to analyze multiple aspects of crossover control simultaneously and rapidly, at high resolution, genome-wide, and on a cell-by-cell basis. Using this approach, we show that loss of interference in zip2 and zip4/spo22 mutants is accompanied by a reduction in crossover homeostasis, thus connecting these two levels of crossover control. We also provide evidence to suggest that repression of crossing over at telomeres and centromeres arises from different mechanisms. Lastly, we uncover a surprising role for the synaptonemal complex component Zip1 in repressing crossing over at the centromere.     

Chromosome 'speed dating' during meiosis of polyploid Brassica hybrids and haploids

Given their tremendous importance for correct chromosome segregation, the number and distribution of crossovers are tightly controlled during meiosis. In this review, we give an overview of crossover formation in polyploid Brassica hybrids and haploids that illustrates or underscores several aspects of crossover control. We first demonstrate that multiple targets for crossover formation (i.e. different but related chromosomes or duplicated regions) are sorted out during meiosis based on their level of relatedness. In euploid Brassica napus (AACC; 2n = 38), crossovers essentially occur between homologous chromosomes and only a few of them form between homeologues. The situation is different in B. napus haploids in which crossovers preferentially occur between homeologous chromosomes and a few can then form between more divergent duplicated regions. We then provide evidence that the frequency of crossovers between a given pair of chromosomes is influenced by the karyotypic and genetic composition of the plants that undergo meiosis. For instance, genetic evidence indicates that the number of crossovers between exactly the same pairs of homologous A chromosomes gets a boost in Brassica digenomic tetraploid (AACC) and triploid (AAC) hybrids. Increased autosyndesis within B. napus haploids as compared to monoploid B. rapa and B. oleracea is another illustration of this process. All these observations may suggest that polyploidization overall boosts up crossover machinery and/or that the number of crossovers is modulated through inter-bivalents or univalent-bivalent cross-talk effects. The last part of this review gives an up-to-date account of what we know about the genetic control of homologous and homeologous crossover formation among Brassica species.     

Meiotic recombination protein Rec12: functional conservation, crossover homeostasis and early crossover/non-crossover decision

In fission yeast and other eukaryotes, Rec12 (Spo11) is thought to catalyze the formation of dsDNA breaks (DSBs) that initiate homologous recombination in meiosis. Rec12 is orthologous to the catalytic subunit of topoisomerase VI (Top6A). Guided by the crystal structure of Top6A, we engineered the rec12 locus to encode Rec12 proteins each with a single amino acid substitution in a conserved residue. Of 21 substitutions, 10 significantly reduced or abolished meiotic DSBs, gene conversion, crossover recombination and the faithful segregation of chromosomes. Critical residues map within the metal ion-binding pocket toprim (E179A, D229A, D231A), catalytic region 5Y-CAP (R94A, D95A, Y98F) and the DNA-binding interface (K201A, G202E, R209A, K242A). A subset of substitutions reduced DSBs but maintained crossovers, demonstrating crossover homeostasis. Furthermore, a strong separation of function mutation (R304A) suggests that the crossover/non-crossover decision is established early by a protein-protein interaction surface of Rec12. Fission yeast has multiple crossovers per bivalent, and chromosome segregation was robust above a threshold of about one crossover per bivalent, below which non-disjunction occurred. These results support structural and functional conservation among Rec12/Spo11/Top6A family members for the catalysis of DSBs, and they reveal how Rec12 regulates other features of meiotic chromosome dynamics.     

Molecular evolution of the meiotic recombination pathway in mammals

Meiotic recombination shapes evolution and helps to ensure proper chromosome segregation in most species that reproduce sexually. Recombination itself evolves, with species showing considerable divergence in the rate of crossing‐over. However, the genetic basis of this divergence is poorly understood. Recombination events are produced via a complicated, but increasingly well‐described, cellular pathway. We apply a phylogenetic comparative approach to a carefully selected panel of genes involved in the processes leading to crossovers—spanning double‐strand break formation, strand invasion, the crossover/non‐crossover decision, and resolution—to reconstruct the evolution of the recombination pathway in eutherian mammals and identify components of the pathway likely to contribute to divergence between species. Eleven recombination genes, predominantly involved in the stabilization of homologous pairing and the crossover/non‐crossover decision, show evidence of rapid evolution and positive selection across mammals. We highlight TEX11 and associated genes involved in the synaptonemal complex and the early stages of the crossover/non‐crossover decision as candidates for the evolution of recombination rate. Evolutionary comparisons to MLH1 count, a surrogate for the number of crossovers, reveal a positive correlation between genome‐wide recombination rate and the rate of evolution at TEX11 across the mammalian phylogeny. Our results illustrate the power of viewing the evolution of recombination from a pathway perspective.     

Differential timing and control of noncrossover and crossover recombination during meiosis.

Unitary models of meiotic recombination postulate that a central intermediate containing Holliday junctions is resolved to generate either noncrossover or crossover recombinants, both of which contain heteroduplex DNA. Contrary to this expectation, we find that during meiosis in Saccharomyces cerevisiae, noncrossover heteroduplex products are formed at the same time as Holliday junction intermediates. Crossovers appear later, when these intermediates are resolved. Furthermore, noncrossover and crossover recombination are regulated differently. ndt80 mutants arrest in meiosis with unresolved Holliday junction intermediates and very few crossovers, while noncrossover heteroduplex products are formed at normal levels and with normal timing. These results suggest that crossovers are formed by resolution of Holliday junction intermediates, while most noncrossover recombinants arise by a different, earlier pathway.     

Analysis of the meiosis in the F1 hybrids of Longiflorum x Asiatic(LA) of lilies (Lilium) using genomic in situ hybridization

Longiflorum and Asiatic lilies of the genus Lilium of the family Liliaeeae are two important groups of modern lily eultivars. One of the main trends of lily breeding is to realize introgression between these groups. With cut style pollination and embryo rescue, distant hybrids between the two groups have been obtained. However, the F1hybrids are highly sterile or some of them could produce a small number of 2n gametes, and their BC1 progenies are usually triploids. Dutch lily breeders have selected many cultivars from these BC1 progenies based on their variation. It is presumably suggested that such variation could be caused by intergenomic recombination and abnormal meiosis during gamete formation in F1 hybrids of Longiflorum x Asiatic (LA) hybrids in Lilium. Therefore, the meiotic process of ten F1 LA hybrids was cytologically investigated using genomic in situ hybridization and traditional cytological methods in the present research.The results showed that: at metaphase I, the homoeologous chromosome pairing among different F1hybrids ranged from 2.0 to 11.4 bi-valents formed by homoeologous chromosomes per pollen mother cell (PMC), and very few multivalents, and even very few bivalents were formed by two chromosomes within one genome rather than homoeologous chromosomes in some PMCs; at anaphase I, all biva-lents were disjoined and most univalents were divided. Both the disjoined bivalents (half-bivalents) and the divided univalents (sister chromatids) moved to the opposite poles, and then formed two groups of chromosomes; because the two resulting half-bivalents retained their axes in the cell undisturbed, many crossover types, including single crossovers, three strand double crossovers, four strand double crossovers, four strand triple crossovers, and four strand multiple crossovers between the non-sister chromatids in the tetrads of bivalents,were clearly inferred by analyzing the breakpoints on the disjoined bivalents. The present investigation not only explained the reason for sterility of the F1 LA hybrids and the variation of their BC1 progenies, but also provided a new method to analyze crossover types in other F1 interspecific hybrids as well.     

Competing crossover pathways act during meiosis in Saccharomyces cerevisiae

In Saccharomyces cerevisiae the MSH4-MSH5, MLH1-MLH3, and MUS81-MMS4 complexes act to promote crossing over during meiosis. MSH4-MSH5, but not MUS81-MMS4, promotes crossovers that display interference. A role for MLH1-MLH3 in crossover control is less clear partly because mlh1Delta mutants retain crossover interference yet display a decrease in crossing over that is only slightly less severe than that seen in msh4Delta and msh5Delta mutants. We analyzed the effects of msh5Delta, mlh1Delta, and mms4Delta single, double, and triple mutants on meiotic crossing over at four consecutive genetic intervals on chromosome XV using newly developed computer software. mlh1Delta mms4Delta double mutants displayed the largest decrease in crossing over (13- to 15-fold) of all mutant combinations, yet these strains displayed relatively high spore viability (42%). In contrast, msh5Delta mms4Delta and msh5Delta mms4Delta mlh1Delta mutants displayed smaller decreases in crossing over (4- to 6-fold); however, spore viability (18-19%) was lower in these strains than in mlh1Delta mms4Delta strains. These data suggest that meiotic crossing over can occur in yeast through three distinct crossover pathways. In one pathway, MUS81-MMS4 promotes interference-independent crossing over; in a second pathway, both MSH4-MSH5 and MLH1-MLH3 promote interference-dependent crossovers. A third pathway, which appears to be repressed by MSH4-MSH5, yields deleterious crossovers.     

Meiotic,genomic and evolutionary properties of crossover distribution in Drosophila yakuba

The number and location of crossovers across genomes are highly regulated during meiosis, yet the key components controlling them are fast evolving, hindering our understanding of the mechanistic causes and evolutionary consequences of changes in crossover rates. Drosophila melanogaster has been a model species to study meiosis for more than a century, with an available high-resolution crossover map that is, nonetheless, missing for closely related species, thus preventing evolutionary context. Here, we applied a novel and highly efficient approach to generate whole-genome high-resolution crossover maps in D. yakuba to tackle multiple questions that benefit from being addressed collectively within an appropriate phylogenetic framework, in our case the D. melanogaster species subgroup. The genotyping of more than 1,600 individual meiotic events allowed us to identify several key distinct properties relative to D. melanogaster. We show that D. yakuba, in addition to higher crossover rates than D. melanogaster, has a stronger centromere effect and crossover assurance than any Drosophila species analyzed to date. We also report the presence of an active crossover-associated meiotic drive mechanism for the X chromosome that results in the preferential inclusion in oocytes of chromatids with crossovers. Our evolutionary and genomic analyses suggest that the genome-wide landscape of crossover rates in D. yakuba has been fairly stable and captures a significant signal of the ancestral crossover landscape for the whole D. melanogaster subgroup, even informative for the D. melanogaster lineage. Contemporary crossover rates in D. melanogaster, on the other hand, do not recapitulate ancestral crossovers landscapes. As a result, the temporal stability of crossover landscapes observed in D. yakuba makes this species an ideal system for applying population genetic models of selection and linkage, given that these models assume temporal constancy in linkage effects. Our studies emphasize the importance of generating multiple high-resolution crossover rate maps within a coherent phylogenetic context to broaden our understanding of crossover control during meiosis and to improve studies on the evolutionary consequences of variable crossover rates across genomes and time.     

Enhanced crossover SCRATCHY: construction and high-throughput screening of a combinatorial library containing multiple non-homologous crossovers

SCRATCHY is a methodology for the construction of libraries of chimeras between genes that display low sequence homology. We have developed a strategy for library creation termed enhanced crossover SCRATCHY, that significantly increases the number of clones containing multiple crossovers. Complementary chimeric gene libraries generated by incremental truncation (ITCHY) of two distinct parental sequences are created, and are then divided into arbitrarily defined sections. The respective sections are amplified by skewed sets of primers (i.e. a combination of gene A specific forward primer and gene B specific reverse primer, etc.) allowing DNA fragments containing non-homologous crossover points to be amplified. The amplified chimeric sections are then subjected to a DNA shuffling process generating an enhanced crossover SCRATCHY library. We have constructed such a library using the rat theta 2 glutathione transferase (rGSTT2) and the human theta 1 glutathione transferase (hGSTT1) genes (63% DNA sequence identity). DNA sequencing analysis of unselected library members revealed a greater diversity than that obtained by canonical family shuffling or with conventional SCRATCHY. Expression and high-throughput flow cytometric screening of the chimeric GST library identified several chimeric progeny that retained rat-like parental substrate specificity.     

Genotype x environment interaction of crossover type: detecting its presence and estimating the crossover point

Genotype-environment interaction (GEI) introduces inconsistency in the relative rating of genotypes across environments and plays a key role in formulating strategies for crop improvement. GEI can be either qualitative (i.e., crossover type) or only quantitative (i.e., non-crossover type). Since the presence of crossover-type interaction has a strong implication for breeding for specific adaptation, it is important to assess the frequency of crossover interactions. This paper presents a test for detecting the presence of crossover-type interaction using the response-environment relationship and enumerates the frequency of crossovers and estimation of the crossover point (CP) on the environment axis, which serves as a cut-off point for the two environments groups where different/specific selections can be made. Sixty-four barley lines with various selection histories were grown in northern Syria and Lebanon giving a total of 21 environments (location-year combinations). Linear regression of the genotypic response on the environmental index represented a satisfactory model, and heterogeneity among regressions was significant. At a 5% level of significance, 38% and 19% of the pairs showed crossover interactions when the error variances were considered heterogeneous and homogeneous, respectively, implying that an appreciable number of crossovers took place in the case of barley lines responding to their environments. The CP of 1.64 t/ha, obtained as the CP of regression lines between the genotype numbers 19 and 31, provided maximum genotype x environment-group interaction. Across all environments, genotype nos. 59 and 12 stood first and second for high yield, respectively. The changes in the ranks of genotypes under the groups of environments can be used for selecting specifically adapted genotypes. Received: 25 January 1999 / Accepted: 16 March 1999     

Chromosome-wide regulation of meiotic crossover formation in Caenorhabditis elegans requires properly assembled chromosome axes

Most sexually reproducing organisms depend on the regulated formation of crossovers, and the consequent chiasmata, to accomplish successful segregation of homologous chromosomes at the meiosis I division. A robust, chromosome-wide crossover control system limits chromosome pairs to one crossover in most meioses in the nematode Caenorhabditis elegans; this system has been proposed to rely on structural integrity of meiotic chromosome axes. Here, we test this hypothesis using a mutant, him-3(me80), that assembles reduced levels of meiosis-specific axis component HIM-3 along cohesin-containing chromosome axes. Whereas pairing, synapsis, and crossing over are eliminated when HIM-3 is absent, the him-3(me80) mutant supports assembly of synaptonemal complex protein SYP-1 along some paired chromosomes, resulting in partial competence for chiasma formation. We present both genetic and cytological evidence indicating that the him-3(me80) mutation leads to an increased incidence of meiotic products with two crossovers. These results indicate that limiting the amount of a major axis component results in a reduced capacity to communicate the presence of a (nascent) crossover and/or to discourage others in response.     

The meiosis-specific zip4 protein regulates crossover distribution by promoting synaptonemal complex formation together with zip2

We have characterized Zip4 (a.k.a. Spo22), a meiosis-specific protein essential for chromosome synapsis in budding yeast. In the absence of Zip4, the synaptonemal complex protein Zip1 fails to polymerize along chromosomes. Zip2 and Zip3 are previously characterized components of the synapsis initiation complex. Zip4 forms a functional unit with Zip2 that is distinct from Zip3. Zip2 and Zip4 are mutually dependent for their chromosomal localization; in polycomplexes, the pattern of Zip2/Zip4 localization is distinct from that of Zip3. Crossing-over is decreased in the zip4 mutant (as in zip1, zip2, and zip3); the remaining crossovers are largely dependent on a parallel pathway utilizing Mms4. zip4 displays a novel phenotype: negative crossover interference, meaning that crossovers tend to cluster. This clustering depends on Zip1. Our results suggest an interaction between crossover pathways such that a protein (Zip1) acting in one pathway influences the distribution of crossovers promoted by a parallel (Mms4-dependent) pathway.     

The absence of crossovers on chromosome 4 in Drosophila melanogaster: Imperfection or interesting exception?

Drosophila melanogaster chromosome 4 is an anomaly because of its small size, chromatin structure, and most notably its lack of crossing over during meiosis. Earlier ideas about the absence of crossovers on 4 hypothesize that these unique characteristics function to prevent crossovers. Here, we explore hypotheses about the absence of crossovers on 4, how these have been addressed, and new insights into the mechanism behind this suppression. We review recently published results that indicate that global crossover patterning, in particular the centromere effect, make a major contribution to the prevention of crossovers on 4.