Germinal Excisions of the Maize Transposon Activator Do Not Stimulate Meiotic Recombination or Homology-Dependent Repair at the Bz Locus

Double-strand breaks have been implicated both in the initiation of meiotic recombination in yeast and as intermediates in the transposition process of nonreplicative transposons. Some transposons of this class, notably P of Drosophila and Tc1 of Caenorhabditis elegans, promote a form of homology-dependent premeiotic gene conversion upon excision. In this work, we have looked for evidence of an interaction between Ac transposition and meiotic recombination at the bz locus in maize. We find that the frequency of meiotic recombination between homologues is not enhanced by the presence of Ac in one of the bz heteroalleles and, conversely, that the presence of a homologous sequence in either trans (homologous chromosome) or cis (tandem duplication) does not promote conversion of the Ac insertion site. However, a tandem duplication of the bz locus may be destabilized by the insertion of Ac. We discuss possible reasons for the lack of interaction between Ac excision and homologous meiotic recombination in maize.     

Extensive interallelic polymorphisms drive meiotic recombination into a crossover pathway

Recombinants isolated from most meiotic intragenic recombination experiments in maize, but not in yeast, are borne principally on crossover chromosomes. This excess of crossovers is not explained readily by the canonical double-strand break repair model of recombination, proposed to account for a large body of yeast data, which predicts that crossovers (COs) and noncrossovers (NCOs) should be recovered equally. An attempt has been made here to identify general rules governing the recovery of the CO and NCO classes of intragenic recombinants in maize. Recombination was analyzed in bz heterozygotes between a variety of mutations derived from the same or different progenitor alleles. The mutations include point mutations, transposon insertions, and transposon excision footprints. Consequently, the differences between the bz heteroalleles ranged from just two nucleotides to many nucleotides, indels, and insertions. In this article, allelic pairs differing at only two positions are referred to as dimorphic to distinguish them from polymorphic pairs, which differ at multiple positions. The present study has revealed the following effects at these bz heteroalleles: (1) recombination between polymorphic heteroalleles produces mostly CO chromosomes; (2) recombination between dimorphic heteroalleles produces both CO and NCO chromosomes, in ratios apparently dependent on the nature of the heteroalleles; and (3) in dimorphic heterozygotes, the two NCO classes are recovered in approximately equal numbers when the two mutations are point mutations but not when one or both mutations are insertions. These observations are discussed in light of a recent version of the double-strand break repair model of recombination that postulates separate pathways for the formation of CO and NCO products.     

CYS3, a hotspot of meiotic recombination in Saccharomyces cerevisiae. Effects of heterozygosity and mismatch repair functions on gene conversion and recombination intermediates

We have examined meiotic recombination at the CYS3 locus. Genetic analysis indicates that CYS3 is a hotspot of meiotic gene conversion, with a putative 5'-3' polarity gradient of conversion frequencies. This gradient is relieved in the presence of msh2 and pms1 mutations, indicating an involvement of mismatch repair functions in meiotic recombination. To investigate the role of mismatch repair proteins in meiotic recombination, we performed a physical analysis of meiotic DNA in wild-type and msh2 pms1 strains in the presence or absence of allelic differences at CYS3. Neither the mutations in CYS3 nor the absence of mismatch repair functions affects the frequency and distribution of nearby recombination-initiating DNA double-strand breaks (DSBs). Processing of DSBs is also similar in msh2 pms1 and wild-type strains. We conclude that mismatch repair functions do not control the distribution of meiotic gene conversion events at the initiating steps. In the MSH2 PMS1 background, strains heteroallelic for frameshift mutations in CYS3 exhibit a frequency of gene conversion greater than that observed for either marker alone. Physical analysis revealed no modification in the formation of DSBs, suggesting that this marker effect results from subsequent processing events that are not yet understood.     

Genetic Fine Structure of the BRONZE Locus in Maize

The bronze (bz) locus in maize, located in the short arm of chromosome 9 (9S), is the structural gene for the anthocyanin biosynthetic enzyme UFGT. The gene has been cloned and its physical map has been oriented relative to the centromere of 9S. We report here the genetic fine structure mapping of several biochemically characterized EMS-induced bz-E mutations, derived from the Bz-W22 isoallele, and Ds insertion bz-m mutations, derived from the Bz-McC isoallele. Two UFGT(-), CRM(+ ) mutants (bz-E2 and bz-E5), which genetically identify coding sequences in the gene, and three UFGT(-), CRM(- )bz-E mutants were mapped against the Ds insertion mutants bz-m1 and bz-m2(DI) by selecting Bz intragenic recombinants from heterozygotes of the type bz-E/bz-m . The exclusive occurrence of one recombinant outside marker class allowed the unambiguous placement of the mutants in a genetic fine structure map. Peculiarly, the two CRM(+)bz-E mutants lie upstream of the three CRM(-)bz-E mutants and at a considerable genetic distance. The UFGT allozymes encoded by the progenitor alleles Bz-W22 and Bz-McC differ in two properties, thermal stability and activity. The sites responsible for these properties were mapped as unselected markers among the Bz intragenic recombinants. The thermal stability site, which also identifies a coding region of the gene, mapped very close to the CRM(+)bz-E mutant sites. The site responsible for variation in activity, which probably identifies a region involved in regulation of expression of the bz locus, mapped at the 5' or proximal end of the locus. It was found to be inseparable from the Ds insertion in bz-m1 that lies very close to the 5' end of the transcribed region.-Evidence was obtained that the insertion of Ds within the bz gene has a suppressing effect on intragenic recombination. Additional data are also presented supporting our observation that Ds affects the pattern of intragenic recombination at bz.-Based on the total genetic length of the bz gene and on the physical size of the transcribed region, we estimate that one unit of recombination at bronze corresponds to 14 kb of DNA. This estimate is more than 100 times smaller than the average value for the whole genome and implies that there may be regions, such as bronze, that serve as hotspots for recombination.     

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.     

Efficient incorporation of large (>2 kb) heterologies into heteroduplex DNA: Pms1/Msh2-dependent and -independent large loop mismatch repair in Saccharomyces cerevisiae

DNA double-strand break (DSB) repair in yeast is effected primarily by gene conversion. Conversion can conceivably result from gap repair or from mismatch repair of heteroduplex DNA (hDNA) in recombination intermediates. Mismatch repair is normally very efficient, but unrepaired mismatches segregate in the next cell division, producing sectored colonies. Conversion of small heterologies (single-base differences or insertions <15 bp) in meiosis and mitosis involves mismatch repair of hDNA. The repair of larger loop mismatches in plasmid substrates or arising by replication slippage is inefficient and/or independent of Pms1p/Msh2p-dependent mismatch repair. However, large insertions convert readily (without sectoring) during meiotic recombination, raising the question of whether large insertions convert by repair of large loop mismatches or by gap repair. We show that insertions of 2.2 and 2.6 kbp convert efficiently during DSB-induced mitotic recombination, primarily by Msh2p- and Pms1p-dependent repair of large loop mismatches. These results support models in which Rad51p readily incorporates large heterologies into hDNA. We also show that large heterologies convert more frequently than small heterologies located the same distance from an initiating DSB and propose that this reflects Msh2-independent large loop-specific mismatch repair biased toward loop loss.     

Analysis of a Recombination Hotspot for Gene Conversion Occurring at the His2 Gene of Saccharomyces Cerevisiae

The properties of gene conversion as measured in fungi that generate asci containing all the products of meiosis imply that meiotic recombination initiates at specific sites. The HIS2 gene of Saccharomyces cerevisiae displays a high frequency of gene conversion, indicating that it is a recombination hotspot. The HIS2 gene was cloned and sequenced, and the cloned DNA was used to make several different types of alterations in the yeast chromosome by transformation; these alterations were used to determine the location of the sequences necessary for the high levels of meiotic conversion observed at HIS2. Previous work indicated that the gene conversion polarity gradient is high at the 3' end of the gene, and that the promoter of the gene is not necessary for the high frequency of conversion observed. Data presented here suggest that at least some of the sequences necessary for high levels of conversion at HIS2 are located over 700 bp downstream of the end of the coding region, extend over (at least) several hundred base pairs, and may be quite complex, perhaps involving chromatin structure. Additional data indicate that multiple single base heterologies within a 1-kb interval contribute little to the frequency of gene conversion. This contrasts with other reports about the role of heterologies at the MAT locus.     

Initiation of meiotic recombination by double-strand DNA breaks in S. pombe

Mitotic gene conversion and reciprocal recombination have recently been shown to be efficiently initiated by double-strand DNA breaks (DSBs) in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. We tested whether DSBs could also initiate meiotic recombination at the mat1 locus in S. pombe. The mat1 switching-mechanism-generated DSB found in mitotically growing cells can be repaired without mat1 switching, since strains deleted for both donor loci (mat2-P and mat3-M) have the break but do not produce inviable cells. A (mat1-P X mat1-M) cross produced a high frequency (20%) of 3:1 gene conversions of mat1 in meiotic tetrads. Gene conversion events were associated with the recombination of flanking markers. Strains lacking the DSB failed to convert. Thus, the DSB at mat1 promotes efficient meiotic recombination in fission yeast.     

Seventeen Complementation Groups of Mutations Decreasing Meiotic Recombination in Schizosaccharomyces Pombe

We have analyzed 43 recessive mutations reducing meiotic intragenic recombination in Schizosaccharomyces pombe. These mutations were isolated by a screen for reduced plasmid-by-chromosome recombination at the ade6 locus. Sixteen of the mutations define 10 new complementation groups, bringing to 17 the number of genes identified to be involved in meiotic recombination. The mutations were grouped into three discrete classes depending on the severity of the recombination deficiency in crosses involving the ade6-M26 recombination hotspot. Class I mutations caused at least a 1000-fold reduction in M26-stimulated intragenic recombination at the ade6 locus. Class II mutations reduced M26-stimulated recombination approximately 100-fold. Class III mutations caused a 3-10-fold reduction in either M26-stimulated or non-hotspot recombination. We obtained multiple alleles of class I and class II mutations, suggesting that we may be nearing saturation for mutations of this type. As a first step toward mapping, we used mitotic segregation to assign fourteen of the rec genes to chromosomes. Mutations in the six rec genes tested also caused a decrease in intragenic recombination at the ura4 locus; five of these mutations also reduced intergenic recombination between the pro2 and arg3 genes. These results indicate that these multiple rec gene products are required for high level meiotic recombination throughout the S. pombe genome.     

Polarity of meiotic gene conversion is 5′ to 3′ within the niaD gene of Aspergillus nidulans

We have examined polarity of meiotic gene conversion in the niiA-niaD gene cluster of Aspergillus nidulans in two-point crosses. The type and position of the mutations represented by the niaD alleles and the correlation between the relative frequency of gene conversion and the physical position of these mutations were determined. We show that polarity of meiotic gene conversion is 5′ to 3′ (transcribed strand) within the niaD gene. Additional crosses involving a niiA allele and a niaD allele show little polarity of gene conversion, which suggests that the recombination events leading to restoration of the niaD gene are initiated upstream of the coding region of the niaD gene but within the niiA-niaD gene cluster, possibly within the intergenic promoter region.     

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.     

Association of reciprocal exchange with gene conversion between the repeated segments of 2-micron circle

The occurrence of reciprocal exchange of flanking DNA during gene conversion between the repeated segments of the yeast plasmid, 2-micron circle has been examined. The conversion event is induced by making a double-stranded gap within one of the repeats in vitro and allowing the gap to be repaired in vivo. The repair takes place with frequent recombination of flanking markers. Neither the topology of the plasmid substrates (linear or circular) nor the relative orientation of the repeats affects the association rule significantly. These events are reminiscent of meiotic gene conversion between homologous chromosomes but contrast sharply with mitotic or meiotic intrachromosomal gene conversion. It would appear that the difference between the outcomes of intramolecular gene conversion on a chromosome and on a plasmid gapped in vitro does not result from the different physical states of intracellular versus transformed DNA. A gene conversion event in a 2-micron circle : : Tn5 plasmid mediated by the 2-micron circle recombinase (FLP) in vivo, which is formally analogous to the yeast mating type interconversion, often results in recombination of flanking markers. The reaction can be mimicked, in the absence of FLP, by gapping the plasmid within one of the 2-micron circle repeats in vitro and carrying out gap repair in vivo.     

Maize genome structure variation: interplay between retrotransposon polymorphisms and genic recombination

Although maize (Zea mays) retrotransposons are recombinationally inert, the highly polymorphic structure of maize haplotypes raises questions regarding the local effect of intergenic retrotransposons on recombination. To examine this effect, we compared recombination in the same genetic interval with and without a large retrotransposon cluster. We used three different bz1 locus haplotypes, McC, B73, and W22, in the same genetic background. We analyzed recombination between the bz1 and stc1 markers in heterozygotes that differ by the presence and absence of a 26-kb intergenic retrotransposon cluster. To facilitate the genetic screen, we used Ds and Ac markers that allowed us to identify recombinants by their seed pigmentation. We sequenced 239 recombination junctions and assigned them to a single nucleotide polymorphism-delimited interval in the region. The genetic distance between the markers was twofold smaller in the presence of the retrotransposon cluster. The reduction was seen in bz1 and stc1, but no recombination occurred in the highly polymorphic intergenic region of either heterozygote. Recombination within genes shuffled flanking retrotransposon clusters, creating new chimeric haplotypes and either contracting or expanding the physical distance between markers. Our findings imply that haplotype structure will profoundly affect the correlation between genetic and physical distance for the same interval in maize.     

Expansions and contractions of the genetic map relative to the physical map of yeast chromosome III.

To examine the relationship between genetic and physical chromosome maps, we constructed a diploid strain of the yeast Saccharomyces cerevisiae heterozygous for 12 restriction site mutations within a 23-kilobase (5-centimorgan) interval of chromosome III. Crossovers were not uniformly distributed along the chromosome, one interval containing significantly more and one interval significantly fewer crossovers than expected. One-third of these crossovers occurred within 6 kilobases of the centromere. Approximately half of the exchanges were associated with gene conversion events. The minimum length of gene conversion tracts varied from 4 base pairs to more than 12 kilobases, and these tracts were nonuniformly distributed along the chromosome. We conclude that the chromosomal sequence or structure has a dramatic effect on meiotic recombination.     

Large Heterologies Impose Their Gene Conversion Pattern onto Closely Linked Point Mutations

We have studied the meiotic non-Mendelian segregation (NMS) pattern of seven large heterologous combinations located in the b2 ascospore gene of Ascobolus. The NMS patterns of these aberration heterozygotes widely differ from each other and from those of point mutations located in the same genetic region. They give lower gene conversion frequencies than point mutations, no postmeiotic segregations (PMS), and either parity or disparity that favors the wild type allele. Two related deletions, G234 and G40, were studied for their effects on the conversion behavior of closely linked point mutations. We found that, when heterozygous, the deletions impose their own NMS pattern onto close mutations. These effects occur on both sides of the heterologies. The effects upon PMS and disparity of linked point mutations gradually disappear as point mutations become more distant. The effects on NMS frequencies and on aberrant 4:4 are polar. They persist for all mutations located downstream from the high conversion end of the gene. This last effect can reflect a blockage of symmetric hDNA formation by large heterologies, whereas the epistasis of the NMS pattern of large heterologies over that of closely linked point mutations suggests that large heterologies and point mutations undergo conversion by means of distinct pathways.     

Suppression of intrachromosomal gene conversion in mammalian cells by small degrees of sequence divergence

Pairs of closely linked defective herpes simplex virus (HSV) thymidine kinase (tk) gene sequences exhibiting various nucleotide heterologies were introduced into the genome of mouse Ltk- cells. Recombination events were recovered by selecting for the correction of a 16-bp insertion mutation in one of the tk sequences. We had previously shown that when two tk sequences shared a region of 232 bp of homology, interruption of the homology by two single nucleotide heterologies placed 19 bp apart reduced recombination nearly 20-fold. We now report that either one of the nucleotide heterologies alone reduces recombination only about 2.5-fold, indicating that the original pair of single nucleotide heterologies acted synergistically to inhibit recombination. We tested a variety of pairs of single nucleotide heterologies and determined that they reduced recombination from 7- to 175-fold. Substrates potentially leading to G-G or C-C mispairs in presumptive heteroduplex DNA (hDNA) intermediates displayed a particularly low rate of recombination. Additional experiments suggested that increased sequence divergence causes a shortening of gene conversion tracts. Collectively, our results suggest that suppression of recombination between diverged sequences is mediated via processing of a mispaired hDNA intermediate.     

Temperature-Sensitive Yeast Mutants Defective in Meiotic Recombination and Replication

A system is described for isolating temperature-sensitive mutants of Saccharomyces cerevisiae with defects in early meiotic events. We used an otherwise haploid strain disomic (n+1) for chromosome III, and heteroallelic at the leucine-2 locus. Meiotic development was initiated by exposure of the strain to acetate sporulation medium, and monitored by the appearance of leucine-independent intragenic recombinants. Mutant isolation was based on the recovery of thermally induced defects in recombination. The temperature-sensitive characteristic was included to allow eventual characterizations of the temporal period during meiosis when each gene performs its essential function. Following mutagenesis with either ethyl methane sulfonate or nitrosoguanidine individual clones were tested at 34° and 24° for acetate-induced recombination. Starting with 2700 clones, derived from cells that survived mutagenic treatment, we isolated 48 strains with thermally induced lesions in recombination. In the majority of mutants premeiotic replication occurred normally, or nearly normally, at the restrictive temperature, indicating that the meiotic cycle was initiated and that there was a defect in an event required for intragenic recombination. We also detected mutants where the thermally induced lesion in recombination resulted from temperature-sensitive premeiotic DNA synthesis.     

Cloning and characterization of DST2, the gene for DNA strand transfer protein beta from Saccharomyces cerevisiae.

The gene encoding the 180-kDa DNA strand transfer protein beta from the yeast Saccharomyces cerevisiae was identified and sequenced. This gene, DST2 (DNA strand transferase 2), was located on chromosome VII. dst2 gene disruption mutants exhibited temperature-sensitive sporulation and a 50% longer generation time during vegetative growth than did the wild type. Spontaneous mitotic recombination in the mutants was reduced severalfold for both intrachromosomal recombination and intragenic gene conversion. The mutants also had reduced levels of the intragenic recombination that is induced during meiosis. Meiotic recombinants were, however, somewhat unstable in the mutants, with a decrease in recombinants and survival upon prolonged incubation in sporulation media. spo13 or spo13 rad50 mutations did not relieve the sporulation defect of dst2 mutations. A dst1 dst2 double mutant has the same phenotype as a dst2 single mutant. All phenotypes associated with the dst2 mutations could be complemented by a plasmid containing DST2.     

Meiotic Recombination-Deficient Mutants of Schizosaccharomyces Pombe

A mutant screen employing the ade6-M26 recombination hotspot was developed and used to isolate Schizosaccharomyces pombe mutants deficient in meiotic recombination. Nine rec mutations were recessive, defining six complementation groups, and reduced ade6 meiotic recombination 3-fold to greater than or equal to 300-fold when homozygous. Three recessive rec mutations analyzed further also reduced meiotic intragenic recombination at ura4 on chromosome III and intergenic recombination between pro2 and arg3 on chromosome I. The observed non-co-ordinate reductions of the recombinant frequencies in the three test intervals suggest a degree of locus (or intragenic vs. intergenic) specificity of the corresponding rec+ gene products. None of the mutations specifically inactivated the ade6-M26 hotspot. Additional rec genes may be identified with these methods.     

Chromosomal Translocations Generated by High-Frequency Meiotic Recombination between Repeated Yeast Genes

We have examined meiotic and mitotic recombination between repeated genes on nonhomologous chromosomes in the yeast Saccharomyces cerevisiae. The results of these experiments can be summarized in three statements. First, gene conversion events between repeats on nonhomologous chromosomes occur frequently in meiosis. The frequency of such conversion events is only 17-fold less than the analogous frequency of conversion between genes at allelic positions on homologous chromosomes. Second, meiotic and mitotic conversion events between repeated genes on nonhomologous chromosomes are associated with reciprocal recombination to the same extent as conversion between allelic sequences. The reciprocal exchanges between the repeated genes result in chromosomal translocations. Finally, recombination between repeated genes on nonhomologous chromosomes occurs much more frequently in meiosis than in mitosis.