Stimulation of Meiotic Recombination in Yeast by an Ars Element

In a previous study, meiotic recombination events were monitored in the 22-kb LEU2 to CEN3 region of chromosome III of Saccharomyces cerevisiae. One region (the hotspot) was shown to have an enhanced level of both gene conversion events and reciprocal crossovers, whereas a second region (the coldspot) was shown to have a depressed level of both types of recombination events. In this study we have analyzed the effects of a replication origin, ARS307, located about 2 kb centromere proximal to the hotspot region, on the distribution of meiotic recombination events. We find that a deletion of this origin results in a reduction of both gene conversions and reciprocal crossovers in the hotspot region, and that a 200-bp fragment of this ARS element can stimulate both types of recombination events when relocated to the coldspot region. Although the magnitude of stimulation of these events is similar in both orientations, whether the ARS is functional or not, the distribution of events is dependent upon the orientation of the element.     

Patterns of heteroduplex formation associated with the initiation of meiotic recombination in the yeast Saccharomyces cerevisiae

The double-strand break repair (DSBR) model of recombination predicts that heteroduplexes will be formed in regions that flank the double-strand break (DSB) site and that the resulting intermediate is resolved to generate either crossovers or noncrossovers for flanking markers. Previous studies in Saccharomyces cerevisiae, however, failed to detect heteroduplexes on both sides of the DSB site. Recent physical studies suggest that some recombination events involve heterodupex formation by a mechanism, synthesis-dependent strand annealing (SDSA), that is inherently asymmetric with respect to the DSB site and that leads exclusively to noncrossovers of flanking markers. Below, we demonstrate that many of the recombination events initiated at the HIS4 recombination hotspot are consistent with a variant of the DSBR model in which the extent of heteroduplex on one side of the DSB site is much greater than that on the other. Events that include only one flanking marker in the heteroduplex (unidirectional events) are usually resolved as noncrossovers, whereas events that include both flanking markers (bidirectional events) are usually resolved as crossovers. The unidirectional events may represent SDSA, consistent with the conclusions of others, although other possibilities are not excluded. We also show that the level of recombination reflects the integration of events initiated at several different DSB sites, and we identify a subset of gene conversion events that may involve break-induced replication (BIR) or repair of a double-stranded DNA gap.     

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.     

Repression of Meiotic Crossing over by a Centromere (CEN3 ) in SACCHAROMYCES CEREVISIAE

The location of the centromere of chromosome III (CEN3) of Saccharomyces cerevisiae has been altered by means of transformation. The frequency of meiotic crossing over in the CEN3-PGK1 and LEU2-CEN3 intervals increases approximately 1.5- and fourfold, respectively, when CEN3 is repositioned at HIS4. The centromere-distal HIS4-LEU2 region experiences a three- to fivefold decrease in the frequency of meiotic exchange when CEN3 is repositioned at HIS4. The inhibition of meiotic crossing over is conferred by a 627-base-pair fragment of CEN3 DNA and is not dependent on the orientation of CEN3 relative to the rest of chromosome III.     

Cloning, Disruption and Chromosomal Mapping of Yeast LEU3 , a Putative Regulatory Gene

The LEU3 gene of the yeast Saccharomyces cerevisiae, which is involved in the regulation of at least two LEU structural genes (LEU1 and LEU2), has been cloned by complementation of leu3 mutations and shown to reside within a 5.6-kb fragment. Transformation of leu3 mutants with LEU3-carrying multicopy plasmids restored normal, leucine-independent growth behavior in the recipients. It also restored approximately wild-type levels of isopropylmalate isomerase (LEU1) and beta-isopropylmalate dehydrogenase (LEU2), which were strongly reduced when exogenous leucine was supplied. Strains containing a disrupted leu3 allele were constructed by deleting 0.7-kb of LEU3 DNA and inserting the yeast HIS3 gene in its place. Like other leu3 mutants, these strains were leaky leucine auxotrophs, owing to a basal level of expression of LEU1 and LEU2. Southern transfer and genetic analyses of strains carrying a disrupted leu3 allele demonstrated that the cloned gene was LEU3, as opposed to a suppressor. Disruption of LEU3 was performed also with a diploid and shown to be nonlethal by tetrad analysis. Northern transfer experiments showed that the LEU3 gene produces mRNA approximately 2.9 kilonucleotides in length. The leu3 marker was mapped to chromosome XII by the spo11 method. Linkage to ura4 by about 44 centiMorgans places leu3 on the right arm of this chromosome.     

A Fine-Structure Map of Spontaneous Mitotic Crossovers in the Yeast Saccharomyces cerevisiae

Homologous recombination is an important mechanism for the repair of DNA damage in mitotically dividing cells. Mitotic crossovers between homologues with heterozygous alleles can produce two homozygous daughter cells (loss of heterozygosity), whereas crossovers between repeated genes on non-homologous chromosomes can result in translocations. Using a genetic system that allows selection of daughter cells that contain the reciprocal products of mitotic crossing over, we mapped crossovers and gene conversion events at a resolution of about 4 kb in a 120-kb region of chromosome V of Saccharomyces cerevisiae. The gene conversion tracts associated with mitotic crossovers are much longer (averaging about 12 kb) than the conversion tracts associated with meiotic recombination and are non-randomly distributed along the chromosome. In addition, about 40% of the conversion events have patterns of marker segregation that are most simply explained as reflecting the repair of a chromosome that was broken in G1 of the cell cycle.     

Construction of a host-vector system in Candida maltosa by using an ARS site isolated from its genome.

To construct a host-vector system in an n-alkane-assimilating yeast, Candida maltosa, the isolation of an ARS site from its genome which replicates autonomously in C. maltosa was attempted. Leu- mutants of C. maltosa were transformed with a gene library prepared by using YEp13 (LEU2+) as a vector, and Leu+ transformants were obtained at a high frequency. A plasmid named pCS1 was isolated from the recipient cells. pCS1 contained a 6.3-kilobase (kb) fragment of the C. maltosa genome, and a 3.8-kb fragment with ARS activity was subcloned and designated the TRA (transformation ability) region. Vectors (pTRA1 and pTRA11) for C. maltosa J288 were constructed that contained this 3.8-kb fragment, pBR322, and the LEU2 gene of Saccharomyces cerevisiae. Transformation of C. maltosa J288 with these plasmids was successful by both spheroplast and lithium acetate methods. Southern blot analysis suggested that the copy number of pTRA1 in C. maltosa was between 10 and 20, and it was stably maintained during growth without selective pressure in the medium. It was also found that these vectors could transform S. cerevisiae leu2- to LEU2+, suggesting that the TRA region contained an ARS site(s) that was specific not only for C. maltosa but also for S. cerevisiae.     

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.     

Sequence non-specific double-strand breaks and interhomolog interactions prior to double-strand break formation at a meiotic recombination hot spot in yeast.

The HIS4LEU2 meiotic recombination hot spot specifies two double-strand break (DSB) sites, I and II. Results presented demonstrate that DSBs at site I occur at many positions throughout a region of approximately 150 bp; we infer that breaks occur in a sequence non-specific fashion. Single-strand nicks at sites I and II are not detectable. Analysis of the effects of a 36 bp linker insertion at site I reveals the existence of communication along and between homologs prior to DSB formation. In cis, the insertion allele causes an increase in DSBs at site I but a decrease in DSBs at site II. In trans, two effects are observed. One effect likely reflects very early pre-DSB interhomolog interactions; the second is suggestive of a later, more intimate interaction in which sites I and II on the two homologs all compete for DSBs. The existence of interhomolog interactions in early meiotic prophase can explain how the sites of crossovers come to lie between the homolog axes at pachytene.     

High-resolution genome-wide analysis of irradiated (UV and γ-rays) diploid yeast cells reveals a high frequency of genomic loss of heterozygosity (LOH) events

In diploid eukaryotes, repair of double-stranded DNA breaks by homologous recombination often leads to loss of heterozygosity (LOH). Most previous studies of mitotic recombination in Saccharomyces cerevisiae have focused on a single chromosome or a single region of one chromosome at which LOH events can be selected. In this study, we used two techniques (single-nucleotide polymorphism microarrays and high-throughput DNA sequencing) to examine genome-wide LOH in a diploid yeast strain at a resolution averaging 1 kb. We examined both selected LOH events on chromosome V and unselected events throughout the genome in untreated cells and in cells treated with either γ-radiation or ultraviolet (UV) radiation. Our analysis shows the following: (1) spontaneous and damage-induced mitotic gene conversion tracts are more than three times larger than meiotic conversion tracts, and conversion tracts associated with crossovers are usually longer and more complex than those unassociated with crossovers; (2) most of the crossovers and conversions reflect the repair of two sister chromatids broken at the same position; and (3) both UV and γ-radiation efficiently induce LOH at doses of radiation that cause no significant loss of viability. Using high-throughput DNA sequencing, we also detected new mutations induced by γ-rays and UV. To our knowledge, our study represents the first high-resolution genome-wide analysis of DNA damage-induced LOH events performed in any eukaryote.     

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.     

Total deletion of yeast LEU4: further evidence for a second alpha-isopropylmalate synthase and evidence for tight LEU4-MET4 linkage

Using a combination of restriction endonuclease digestion, nuclease BAL 31 treatment, and standard ligation procedures, a 4.4-kb DNA segment that carried the yeast LEU4 gene [encoding alpha-isopropylmalate synthase (IPMS) I] and adjoining sequences was excised from an appropriate plasmid and replaced with the yeast HIS3 gene. The new plasmid was digested to obtain a linear HIS3-carrying fragment flanked by remnants of the LEU4 region. Integrative transformation of a LEU4fbr LEU5+ his3- strain with this fragment resulted in the deletion of the LEU4 gene from the genome of some recipients, as demonstrated by transformant phenotype, genetic analysis and the absence of RNA capable of hybridizing to a LEU4 probe. The leu4 deletion strains remained Leu+. The extract of one such strain contained about 18% of the IPMS activity of wild-type cells. It is concluded that the residual activity is that of a second IPMS (IPMS II) that depends on an intact LEU5 locus. IPMS II was inhibited by leucine, but its sensitivity was about an order of magnitude lower than that of IPMS I. Deletion of the LEU4 region by the method utilized here resulted in an amino acid auxotrophy that could be satisfied by methionine, homocysteine, or cysteine. Complementation tests and genetic analysis demonstrated that the affected gene was MET4. Linkage to MET4 would place the LEU4 gene on the left arm of chromosome XIV.     

Telomeric site position heterogeneity in macronuclear DNA of Paramecium primaurelia.

In Paramecium primaurelia, the macronuclear gene encoding the G surface protein is located near a telomere. In this study, multiple copies of this telomere have been isolated and the subtelomeric and telomeric regions of some of them have been sequenced. The telomeric sequences consist of tandem repeats of the hexanucleotides C4A2 or C3A3. We show that the location where these repeats are added, which we call the telomeric site, is variable within a 0.6-0.8-kb region. These results are discussed in relation with the formation of macronuclear DNA.     

Reciprocal crossovers and a positional preference for strand exchange in recombination events resulting in deletion or duplication of chromosome 17p11.2

Smith-Magenis syndrome (SMS) is caused by an approximately 4-Mb heterozygous interstitial deletion on chromosome 17p11.2 in approximately 80%-90% of affected patients. Three large ( approximately 200 kb), complex, and highly homologous ( approximately 98%) low-copy repeats (LCRs) are located inside or flanking the SMS common deletion. These repeats, also known as "SMS-REPs," are termed "distal," "middle," and "proximal." The directly oriented distal and proximal copies act as substrates for nonallelic homologous recombination resulting in both the deletion associated with SMS and the reciprocal duplication: dup(17)(p11.2p11.2). Using restriction enzyme cis-morphism analyses and direct sequencing, we mapped the regions of strand exchange in 16 somatic-cell hybrids that harbor only the recombinant SMS-REP. Our studies showed that the sites of crossovers were distributed throughout the region of homology between the distal and proximal SMS-REPs. However, despite approximately 170 kb of high homology, 50% of the recombinant junctions occurred in a 12.0-kb region within the KER gene clusters. DNA sequencing of this hotspot (positional preference for strand exchange) in seven recombinant SMS-REPs narrowed the crossovers to an approximately 8-kb interval. Four of them occurred in a 1,655-bp region rich in polymorphic nucleotides that could potentially reflect frequent gene conversion. For further evaluation of the strand exchange frequency in patients with SMS, novel junction fragments from the recombinant SMS-REPs were identified. As predicted by the reciprocal-recombination model, junction fragments were also identified from this hotspot region in patients with dup(17)(p11.2p11.2), documenting reciprocity of the positional preference for strand exchange. Several potential cis-acting recombination-promoting sequences were identified within the hotspot. It is interesting that we found 2.1-kb AT-rich inverted repeats flanking the proximal and middle KER gene clusters but not the distal one. The role of any or all of these in stimulating double-strand breaks around this positional recombination hotspot remains to be explored.