The M26 hotspot of Schizosaccharomyces pombe stimulates meiotic ectopic recombination and chromosomal rearrangements.

Homologous recombination is increased during meiosis between DNA sequences at the same chromosomal position (allelic recombination) and at different chromosomal positions (ectopic recombination). Recombination hotspots are important elements in controlling meiotic allelic recombination. We have used artificially dispersed copies of the ade6 gene in Schizosaccharomyces pombe to study hotspot activity in meiotic ectopic recombination. Ectopic recombination was reduced 10-1000-fold relative to allelic recombination, and was similar to the low frequency of ectopic recombination between naturally repeated sequences in S. pombe. The M26 hotspot was active in ectopic recombination in some, but not all, integration sites, with the same pattern of activity and inactivity in ectopic and allelic recombination. Crossing over in ectopic recombination, resulting in chromosomal rearrangements, was associated with 35-60% of recombination events and was stimulated 12-fold by M26. These results suggest overlap in the mechanisms of ectopic and allelic recombination and indicate that hotspots can stimulate chromosomal rearrangements.     

Meiotic Gene Conversion and Crossing over between Dispersed Homologous Sequences Occurs Frequently in Saccharomyces cerevisiae

We have examined meiotic recombination between two defined leu2 heteroalleles present at the normal LEU2 locus and in leu2-containing plasmids inserted at four other genomic locations. In diploids where the two leu2 markers were present at allelic locations on parental homologs, the frequency of Leu2+ spores varied 38-fold, in a location-dependent manner. These results indicate that recombination in a genetic interval can be modulated by sequences at least 2.7 kb outside that interval. Leu2+ meiotic segregants were also recovered from diploids where LEU2 was marked with one heteroallele, and the other leu2 heteroallele was inserted at another genomic location. These products of ectopic interactions, between dispersed copies of leu2 sharing only 2.2 kb of homology, were recovered at a frequency comparable to that observed in corresponding allelic crosses. This high frequency of ectopic meiotic recombination was observed in crosses where both recombining partners could potentially pair with sequences at an allelic position. In addition, a significant fraction (22-50%) of these ectopic recombinants were associated with crossing over of flanking sequences.     

The Ade6-M26 Mutation of Schizosaccharomyces Pombe Increases the Frequency of Crossing over

The ade6-M26 mutation of Schizosaccharomyces pombe increases conversion frequency in comparison with the nearby mutation ade6-M375. In order to investigate the effect of ade6-M26 on crossover frequency, heteroallelic ade6 duplications were constructed by integration of plasmids carrying the marker gene ura4. One ade6 gene carries either of the mutations M26 or M375 while the other ade6 copy carries the L469 mutation in both duplications. The duplication with ade6-M26 yields Ade(+) recombinants at significantly higher frequencies in meiosis, but not in mitosis. Tetrad analysis and physical characterization of spore clones from recombination tetrads demonstrate that conversions, unequal crossovers and intrachromatid exchanges occur at higher frequencies but with unaltered proportions among them. The conversion events show a pronounced bias when M26 is involved: they take place preferentially at the M26 allele. Thus the ade6-M26 mutation not only enhances conversion frequency as demonstrated before, but also crossover frequency. It displays the properties expected for a preferred site of initiation of general meiotic recombination. The duplications also yielded new information on ectopic recombination in S. pombe: ectopic crossovers occur in the duplications at much higher frequency than among naturally dispersed homologous sequences.     

A natural meiotic DNA break site in Schizosaccharomyces pombe is a hotspot of gene conversion, highly associated with crossing over

In Schizosaccharomyces pombe, meiosis-specific DNA breaks that initiate recombination are observed at prominent but widely separated sites. We investigated the relationship between breakage and recombination at one of these sites, the mbs1 locus on chromosome I. Breaks corresponding to 10% of chromatids were mapped to four clusters spread over a 2.1-kb region. Gene conversion of markers within the clusters occurred in 11% of tetrads (3% of meiotic chromatids), making mbs1 a conversion hotspot when compared to other fission yeast markers. Approximately 80% of these conversions were associated with crossing over of flanking markers, suggesting a strong bias in meiotic break repair toward the generation of crossovers. This bias was observed in conversion events at three other loci, ade6, ade7, and ura1. A total of 50-80% of all crossovers seen in a 90-kb region flanking mbs1 occurred in a 4.8-kb interval containing the break sites. Thus, mbs1 is also a hotspot of crossing over, with breakage at mbs1 generating most of the crossovers in the 90-kb interval. Neither Rec12 (Spo11 ortholog) nor I-SceI-induced breakage at mbs1 was significantly associated with crossing over in an apparently break-free interval >25 kb away. Possible mechanisms for generating crossovers in such break-free intervals are discussed.     

Effects of Mutagen-Sensitive Mus Mutations on Spontaneous Mitotic Recombination in Aspergillus

Methyl methane-sulfonate (MMS)-sensitive, radiation-induced mutants of Aspergillus were shown to define nine new DNA repair genes, musK to musS. To test mus mutations for effects on mitotic recombination, intergenic crossing over was assayed between color markers and their centromeres, and intragenic recombination between two distinguishable adE alleles. Of eight mutants analyzed, four showed significant deviations from mus+ controls in both tests. Two mutations, musK and musL, reduced recombination, while musN and musQ caused increases. In contrast, musO diploids produced significantly higher levels only for intragenic recombination. Effects were relatively small, but averages between hypo- and hyperrec mus differed 15-20-fold. In musL diploids, most of the rare color segregants resulted from mitotic malsegregation rather than intergenic crossing over. This indicates that the musL gene product is required for recombination and that DNA lesions lead to chromosome loss when it is deficient. In addition, analysis of the genotypes of intragenic (ad+) recombinants showed that the musL mutation specifically reduced single allele conversion but increased complex conversion types (especially recombinants homozygous for ad+). Similar analysis revealed differences between the effects of two hyperrec mutations; musN apparently caused high levels solely of mitotic crossing over, while musQ increased various conversion types but not reciprocal crossovers. These results suggest that mitotic gene conversion and crossing over, while generally associated, are affected differentially in some of the mus strains of Aspergillus nidulans.     

Position Effects in Ectopic and Allelic Mitotic Recombination in Saccharomyces Cerevisiae

We have examined the role that genomic location plays in mitotic intragenic recombination. Mutant alleles of the LEU2 gene were inserted at five locations in the yeast genome. Diploid and haploid strains containing various combinations of these inserts were used to examine both allelic recombination (between sequences at the same position on parental homologs) and ectopic recombination (between sequences at nonallelic locations). Chromosomal location had little effect on mitotic allelic recombination. The rate of recombination to LEU2 at five different loci varied less than threefold. This finding contrasts with previous observations of strong position effects in meiosis; frequencies of meiotic recombination at the same five loci differ by about a factor of forty. Mitotic recombination between dispersed copies of leu2 displayed strong position effects. Copies of leu2 located approximately 20 kb apart on the same chromosome recombined at rates 6-13-fold higher than those observed for allelic copies of leu2. leu2 sequences located on nonhomologous chromosomes or at distant loci on the same chromosome recombined at rates similar to those observed for allelic copies. We suggest that, during mitosis, parental homologs interact with each other no more frequently than do nonhomologous chromosomes.     

Mismatch Repair-Induced Meiotic Recombination Requires the Pms1 Gene Product

The presence of multiple heterologies in a 9-kilobase (kb) interval results in a decrease in meiotic crossovers from 26.0% to 10.1%. There is also an increase from 3.5% to 11.1% in gene conversions and ectopic recombinations between the flanking homologous MAT loci. The hypothesis that mismatch repair of heteroduplex DNA containing several heterologies would lead to a second round of recombination has now been tested by examining the effect of a mutation that reduces mismatch correction. The repair-defective pms1-1 allele restores the pattern of recombination to nearly that seen in congenic diploids without the heterologies. Mismatch repair-induced recombination causes a significant increase in MAT conversions and ectopic recombination events with as few as two heterozygosities separated by 0.3-0.7 kb, but not when the mismatches are separated by greater than 1 kb. The frequency of these events depends on both the number and position of the heterozygosities relative to the flanking homologous MAT loci used to detect the events. The creation of recombinogenic lesions by mismatch repair in yeast could be analogous to the creation of recombinogenic lesions in dam- Escherichia coli. We suggest that the repair of heteroduplex DNA containing multiple mismatches may produce chromosomal rearrangements and gamete inviability when naturally polymorphic chromosomes undergo meiotic recombination.     

The Efficiency of Meiotic Recombination between Dispersed Sequences in Saccharomyces Cerevisiae Depends upon Their Chromosomal Location

To examine constraints imposed on meiotic recombination by homologue pairing, we measured the frequency of recombination between mutant alleles of the ARG4 gene contained in pBR322-based inserts. Inserts were located at identical loci on homologues (allelic recombination) or at different loci on either homologous or heterologous chromosomes (ectopic recombination). Ectopic recombination between interstitially located inserts on heterologous chromosomes had an efficiency of 6-12% compared to allelic recombination. By contrast, ectopic recombination between interstitial inserts located on homologues had relative efficiencies of 47-99%. These findings suggest that when meiotic ectopic recombination occurs, homologous chromosomes are already colocalized. The efficiency of ectopic recombination between inserts on homologues decreased as the physical distance between insert sites was increased. This result is consistent with the suggestion that during meiotic recombination, homologues are not only close to each other, but also are aligned end to end. Finally, the efficiency of ectopic recombination between inserts near telomeres (within 16 kb) was significantly greater than that observed with inserts >50 kb from the nearest telomere. Thus, at the time of recombination, there may be a special relationship between the ends of chromosomes not shared with interstitial regions.     

Contractions and expansions of CAG/CTG trinucleotide repeats occur during ectopic gene conversion in yeast,by a MUS81-independent mechanism

CAG/CTG trinucleotide repeat tracts expand and contract at a high rate during gene conversion in Saccharomyces cerevisiae. In order to characterize the mechanism responsible for such rearrangements, we built an experimental system based on the use of the rare cutter endonuclease I-SceI, to study the fate of trinucleotide repeat tracts during meiotic or mitotic (allelic or ectopic) gene conversion. After double-strand break (DSB) induced meiotic recombination, (CAG)(98) and (CAG)(255) are rearranged in 5% and 52% of the gene conversions, respectively, with similar proportions of contractions and expansions. No evidence of a meiotic hot spot activity associated with trinucleotide repeats could be found. When gene conversion is induced by a DSB during mitotic growth of the cells, no rearrangement of the repeat tracts is detected when the donor sequence is allelic to the recipient site of the DSB. However, when the donor sequence is at an ectopic location, frequent contractions and expansions of the repeat tract are found. No crossing-over associated with gene conversion could be detected. Mutants for the MUS81 gene, involved in the resolution of recombination intermediates, show a frequency of rearrangements identical with that of the wild-type strain. We concluded that trinucleotide repeat rearrangements occur frequently during ectopic but not during allelic recombination, by a mechanism that does not require crossover formation.     

Meiotic Crossing over between Nonhomologous Chromosomes Affects Chromosome Segregation in Yeast

Meiotic recombination between artificial repeats positioned on nonhomologous chromosomes occurs efficiently in the yeast Saccharomyces cerevisiae. Both gene conversion and crossover events have been observed, with crossovers yielding reciprocal translocations. In the current study, 5.5-kb ura3 repeats positioned on chromosomes V and XV were used to examine the effect of ectopic recombination on meiotic chromosome segregation. Ura(+) random spores were selected and gene conversion vs. crossover events were distinguished by Southern blot analysis. Approximately 15% of the crossover events between chromosomes V and XV were associated with missegregation of one of these chromosomes. The missegregation was manifest as hyperploid spores containing either both translocations plus a normal chromosome, or both normal chromosomes plus one of the translocations. In those cases where it could be analyzed, missegregation occurred at the first meiotic division. These data are discussed in terms of a model in which ectopic crossovers compete efficiently with normal allelic crossovers in directing meiotic chromosome segregation.     

Genetic Analysis of the Reciprocal Translocation T2(I;VIII) of Aspergillus Using the Technique of Mitotic Mapping in Homozygous Translocation Diploids

A UV-induced sulphite-requiring mutant (sD50) consistently shows mitotic linkage to groups I and VIII in haploids from heterozygous mapping diploids. This linkage was found to be due to a reciprocal translocation T2(I;VIII) which could not be separated from the sulphite requirement in about 100 tested progeny from heterozygous crosses, and both may well have been induced by the same mutational event. T2(I;VIII) is the first case of a reciprocal translocation in Aspergillus which showed meiotic linkages between markers of different linkage groups, and, in addition, involved chromosome arms containing markers suitable for complete mapping by the technique of mitotic recombination in homozygous translocation diploids.-Using various selective markers, haploid segregants and diploid crossovers of all possible types were isolated from homozygous translocation diploids. (1) Haploid segregants showed new linkage relationships in T/T diploids: all available markers of VIII now segregated as a group with the majority of the markers of I, except for the markers of the left tip of I. These formed a separate linkage group and are presumably translocated to VIII. (2) Diploid mitotic crossovers confirmed this information and showed that the orientation of the translocated segments was unchanged. These findings conclusively demonstrate that T2(I;VIII) is a reciprocal translocation due to an exchange of the left tip of group I with the long right arm of group VIII.-Since the position of the break on VIIIR was found to be at sD50 this marker could be used to map the break on IL by meiotic recombination in heterozygous crosses. In addition, such crosses showed reduced recombination around the breaks, so that it was possible to sequence markers which normally are barely linked.     

Meiotic exchange within and between chromosomes requires a common Rec function in Saccharomyces cerevisiae.

We used haploid yeast cells that express both the MATa and MAT alpha mating-type alleles and contain the spo13-1 mutation to characterize meiotic recombination within single, unpaired chromosomes in Rec+ and Rec- Saccharomyces cerevisiae. In Rec+ haploids, as in diploids, intrachromosomal recombination in the ribosomal DNA was detected in 2 to 6% of meiotic divisions, and most events were unequal reciprocal sister chromatid exchange (SCE). By contrast, intrachromosomal recombination between duplicated copies of the his4 locus occurred in approximately 30% of haploid meiotic divisions, a frequency much higher than that reported in diploids; only about one-half of the events were unequal reciprocal SCE. The spo11-1 mutation, which virtually eliminates meiotic exchange between homologs in diploid meiosis, reduced the frequency of intrachromosomal recombination in both the ribosomal DNA and the his4 duplication during meiosis by 10- to greater than 50-fold. This Rec- mutation affected all forms of recombination within chromosomes: unequal reciprocal SCE, reciprocal intrachromatid exchange, and gene conversion. Intrachromosomal recombination in spo11-1 haploids was restored by transformation with a plasmid containing the wild-type SPO11 gene. Mitotic intrachromosomal recombination frequencies were unaffected by spo11-1. This is the first demonstration of a gene product required for recombination between homologs as well as recombination within chromosomes during meiosis.     

OsHUS1 Facilitates Accurate Meiotic Recombination in Rice

Meiotic recombination normally takes place between allelic sequences on homologs. This process can also occur between non-allelic homologous sequences. Such ectopic interaction events can lead to chromosome rearrangements and are normally avoided. However, much remains unknown about how these ectopic interaction events are sensed and eliminated. In this study, using a screen in rice, we characterized a homolog of HUS1 and explored its function in meiotic recombination. In Oshus1 mutants, in conjunction with nearly normal homologous pairing and synapsis, vigorous, aberrant ectopic interactions occurred between nonhomologous chromosomes, leading to multivalent formation and subsequent chromosome fragmentation. These ectopic interactions relied on programed meiotic double strand breaks and were formed in a manner independent of the OsMER3-mediated interference-sensitive crossover pathway. Although early homologous recombination events occurred normally, the number of interference-sensitive crossovers was reduced in the absence of OsHUS1. Together, our results indicate that OsHUS1 might be involved in regulating ectopic interactions during meiosis, probably by forming the canonical RAD9-RAD1-HUS1 (9-1-1) complex.     

UV-Induced mitotic recombination and its dependence on photoreactivation and liquid holding in the rad6-1 mutant of Saccharomyces cerevisiae

Summary Spontaneous and UV-induced mitotic recombination was compared in diploids homozygous for rad6-1 mutation and in the wild-type strain carrying heterozygous markers for detecting gene conversion (hom2-1, hom2-2) and crossing over (adel, ade2). Diploids homozygous for rad6-1 mutation were characterised by an elevated level of spontaneous and UV-induced mitotic recombination, particularly the intergenic events. Exposure of UV-irradiated strains to visible light resulted in an increased survival and decreased level of mitotic recombination. Liquid holding (LH) differentially affected frequency of mitotic intergenic and intragenic recombination in mutant and wild-type strains, being without any significant effect on cell survival. In a mutant strain intragenic recombination is significantly increased, intergenic only slightly. In the wild-type strain intragenic recombination is slightly decreased but intergenic is not changed by LH. Visible light applied after LH had no effect on survival and mitotic recombination in the wild type, while in the mutant strain photoreactivability of survival was fully preserved and accompanied by a decrease in the frequency of intragenic and intergenic recombination. The results suggest that metabolic pathways responsible for restoring cell survival are independent of or only partly overlapping with those concerning recombination events.     

Expression of Saccharomyces cerevisiae MATa and MAT alpha enhances the HO endonuclease-stimulation of chromosomal rearrangements directed by his3 recombinational substrates

Radiation resistance in Saccharomyces cerevisiae is greater in a/alpha diploids than in aa or alpha alpha diploids, and higher levels of radiation resistance correlates with more mitotic recombination. Specifically, we investigated whether the stimulation of directed translocations, inversions, and unequal sister chromatid exchanges (SCEs) by HO endonuclease-induced double-strand breaks (DSBs) is enhanced in a/alpha cells. These rearrangements result from mitotic recombination between two truncated his3 genes, his3-delta 5' and his3-delta 3'::HOcs, positioned on non-homologous chromosomes or positioned in juxtaposition on the same chromosome in inverted or direct orientation. Mitotic recombination was initiated by HO endonuclease-induced DSBs at the HO cut site (HOcs) located at his3-delta 3'::HOcs, and His+ recombinants were selected. In MATa-inc haploid strains, which do not switch mating-type, the DSB reduced viability, relative to undamaged cells, and increases the frequency of His+ recombinants containing translocations to 2.4 x 10(-4) (seven-fold), SCEs to 5.4 x 10(-4) (five-fold), and inversions to 1.8 x 10(-3) (six-fold). Compared to a haploids, DSB-stimulated frequencies in a/alpha haploids were three-fold higher for translocations, two-fold higher for SCEs, and ten-fold higher for inversions; however DSB-induced lethality was greater in a/alpha haploids. Compared to aa diploids, DSB-stimulated frequencies of translocations and viability after chromosome cleavage were greater in a/alpha diploids. We suggest that heterozygosity at MAT may elevate the frequency of DSB-initiated reciprocal exchange events in both haploid and diploid cells, but may only increase viability after chromosome cleavage in diploid cells.     

The nature and extent of synaptonemal complex formation in haploid barley

Normal synaptonemal complexes have been found in haploid barley meiotic prophase at stages equivalent to pachytene in diploids. Reconstructions of serially sectioned nuclei have shown that up to 60% of the haploid chromosomes may pair in either intra- or interchromosomal associations. The extent and nature of the synaptonemal complex formation suggest that the chromosome pairing is non-homologous. From the virtual absence of chiasmata in metaphase I stages of the haploids it is inferred that crossing over requires a more precise DNA alignment than is provided by synaptonemal complex formation alone.     

mus309 mutation, defective in DNA double-strand break repair, affects intergenic but not intragenic meiotic recombination in Drosophila melanogaster

The effect was investigated of the hypomorphic DNA double-strand break repair, notably synthesis-dependent strand annealing, deficient mutation mus309 on the third chromosome of Drosophila melanogaster on intergenic and intragenic meiotic recombination in the X chromosome. The results showed that the mutation significantly increases the frequency of intergenic crossing over in two of three gene intervals of the X chromosome studied. Interestingly the increase was most prevalent in the tip of the X chromosome where crossovers normally are least frequent per physical map unit length. In particular crossing over interference was also affected, indicating that the effect of the mus309 mutation involves preconditions of crossing over but not the event of crossing over itself. On the other hand, the results also show that most probably the mutation does not have any effect on intragenic recombination, i.e. gene conversion. These results are fully consistent with the present molecular models of meiotic crossing over initiated by double-strand breaks of DNA followed by formation of a single-end-invasion intermediate, or D-loop, which is subsequently processed to generate either crossover or non-crossover products involving formation of a double Holliday junction. In particular the results suggest that the mus309 gene is involved in resolution of the D-loop, thereby affecting the choice between double-strand-break repair (DSBR) and synthesis-dependent strand annealing (SDSA) pathways of meiotic recombination.     

Meiotic recombination at the ends of chromosomes in Saccharomyces cerevisiae

Meiotic reciprocal recombination (crossing over) was examined in the outermost 60-80 kb of almost all Saccharomyces cerevisiae chromosomes. These sequences included both repetitive gene-poor subtelomeric heterochromatin-like regions and their adjacent unique gene-rich euchromatin-like regions. Subtelomeric sequences underwent very little crossing over, exhibiting approximately two- to threefold fewer crossovers per kilobase of DNA than the genomic average. Surprisingly, the adjacent euchromatic regions underwent crossing over at twice the average genomic rate and contained at least nine new recombination "hot spots." These results prompted an analysis of existing genetic mapping data, which showed that meiotic reciprocal recombination rates were on average greater near chromosome ends exclusive of the subtelomeres. Thus, the distribution of crossovers in S. cerevisiae appears to resemble that found in several higher eukaryotes where the outermost chromosomal regions show increased crossing over.     

The meiotic bouquet promotes homolog interactions and restricts ectopic recombination in Schizosaccharomyces pombe

Chromosome architecture undergoes extensive, programmed changes as cells enter meiosis. A highly conserved change is the clustering of telomeres at the nuclear periphery to form the "bouquet" configuration. In the fission yeast Schizosaccharomyces pombe the bouquet and associated nuclear movement facilitate initial interactions between homologs. We show that Bqt2, a meiosis-specific protein required for bouquet formation, is required for wild-type levels of homolog pairing and meiotic allelic recombination. Both gene conversion and crossing over are reduced and exhibit negative interference in bqt2Delta mutants, reflecting reduced homolog pairing. While both the bouquet and nuclear movement promote pairing, only the bouquet restricts ectopic recombination (that between dispersed repetitive DNA). We discuss mechanisms by which the bouquet may prevent deleterious translocations by restricting ectopic recombination.     

The role of centromere alignment in meiosis I segregation of homologous chromosomes in Saccharomyces cerevisiae

During meiosis, homologous chromosomes pair and then segregate from each other at the first meiotic division. Homologous centromeres appear to be aligned when chromosomes are paired. The role of centromere alignment in meiotic chromosome segregation was investigated in Saccharomyces cerevisiae diploids that contained one intact copy of chromosome I and one copy bisected into two functional centromere-containing fragments. The centromere on one fragment was aligned with the centromere on the intact chromosome while the centromere on the other fragment was either aligned or misaligned. Fragments containing aligned centromeres segregated efficiently from the intact chromosome, while fragments containing misaligned centromeres segregated much less efficiently from the intact chromosome. Less efficient segregation was correlated with crossing over in the region between the misaligned centromeres. Models that suggest that these crossovers impede proper segregation by preventing either a segregation-promoting chromosome alignment on the meiotic spindle or some physical interaction between homologous centromeres are proposed.