Meiotic recombination between repeated transposable elements in Saccharomyces cerevisiae.

We have measured the frequency of meiotic recombination between marked Ty elements in the Saccharomyces cerevisiae genome. These recombination events were usually nonreciprocal (gene conversions) and sometimes involved nonhomologous chromosomes. The frequency of ectopic gene conversion among Ty elements appeared lower than expected on the basis of previous studies of recombination between artificially constructed repeats. The conversion events involved either a subset of the total Ty elements in the genome or the conversion tract was restricted to a small region of the Ty element. In addition, the observed conversion events were very infrequently associated with reciprocal exchange.     

Intrachromosomal movement of genetically marked Saccharomyces cerevisiae transposons by gene conversion.

In this paper, we describe the movement of a genetically marked Saccharomyces cerevisiae transposon. Ty912(URA3), to new sites in the S. cerevisiae genome. Ty912 is an element present at the HIS4 locus in the his4-912 mutant. To detect movement of Ty912, this element has been genetically marked with the S. cerevisiae URA3 gene. Movement of Ty912(URA3) occurs by recombination between the marked element and homologous Ty elements elsewhere in the S. cerevisiae genome. Ty912(URA3) recombines most often with elements near the HIS4 locus on chromosome III, less often with Ty elements elsewhere on chromosome III, and least often with Ty elements on other chromosomes. These recombination events result in changes in the number of Ty elements present in the cell and in duplications and deletions of unique sequence DNA.     

Deletions extending from a single Ty1 element in Saccharomyces cerevisiae.

Chromosomal rearrangements associated with one Ty1 element in the iso-1-cytochrome c (CYC1) region of Saccharomyces cerevisiae yeast cells were examined. Most of the rearrangements were deletions of the three linked genes, CYC1, OSM1, and RAD7, and resulted from recombination involving the single Ty1 element and a solo delta in the same orientation. These deletions differed by the number of Ty1 elements (zero, one, or two) remaining after deletion and by restriction site heterogeneities associated with these elements. A single Ty1 element remained at the deletion junction point much more frequently than no Ty1. Apparently the Ty1-associated delta element nearer to the solo delta was involved more often in recombination than the more distal Ty1-associated delta element. The restriction site data implicate gene conversion and suggest that site-specific recombination within the deltas, if occurring, is not the only mechanism of delta-delta recombination. Three other rearrangements bore deletions which began at the end of the Ty1 element and extended into regions not bearing Ty1 or delta sequences. Two of these deletions eliminated 7 kilobases of DNA, although they differed by an associated reciprocal translocation. The third involved a deletion of 14.7 kilobases of DNA associated with an overlapping inversion.     

Ty1 copy number dynamics in Saccharomyces

To understand long terminal repeat (LTR)-retrotransposon copy number dynamics, Ty1 elements were reintroduced into a "Ty-less" Saccharomyces strain where elements had been lost by LTR-LTR recombination. Repopulated strains exhibited alterations in chromosome size that were associated with Ty1 insertions, but did not become genetically isolated. The rates of element gain and loss under genetic and environmental conditions known to affect Ty1 retrotransposition were determined using genetically tagged reference elements. The results show that Ty1 retrotransposition varies with copy number, temperature, and cell type. In contrast to retrotransposition, Ty1 loss by LTR-LTR recombination was more constant and not markedly influenced by copy number. Endogenous Ty1 cDNA was poorly utilized for recombination when compared with LTR-LTR recombination or ectopic gene conversion. Ty1 elements also appear to be more susceptible to copy number fluctuation in haploid cells. Ty1 gain/loss ratios obtained under different conditions suggest that copy number oscillates over time by altering the rate of retrotransposition, resulting in the diverse copy numbers observed in Saccharomyces.     

cDNA of the Yeast Retrotransposon Ty5 Preferentially Recombines with Substrates in Silent Chromatin

The yeast retrotransposon Ty5 preferentially integrates into regions of silent chromatin. Ty5 cDNA also recombines with homologous sequences, generating tandem elements or elements that have exchanged markers between cDNA and substrate. In this study, we demonstrate that Ty5 integration depends upon the conserved DD(35)E domain of integrase and cis-acting sequences at the end of the long terminal repeat (LTR) implicated in integrase binding. cDNA recombination requires Rad52p, which is responsible for homologous recombination. Interestingly, Ty5 cDNA recombines at least three times more frequently with substrates in silent chromatin than with a control substrate at an internal chromosomal locus. This preference depends upon the Ty5 targeting domain that is responsible for integration specificity, suggesting that localization of cDNA to silent chromatin results in the enhanced recombination. Recombination with a telomeric substrate occasionally generates highly reiterated Ty5 arrays, and mechanisms for tandem element formation were explored by using a plasmid-based recombination assay. Point mutations were introduced into plasmid targets, and recombination products were characterized to determine recombination initiation sites. Despite our previous observation of the importance of the LTR in forming tandem elements, recombination cannot simply be explained by crossover events between the LTRs of substrate and cDNA. We propose an alternative model based on single-strand annealing, where single-stranded cDNA initiates tandem element formation and the LTR is required for strand displacement to form a looped intermediate. Retrotransposons are increasingly found associated with chromosome ends, and amplification of Ty5 by both integration and recombination exemplifies how retroelements can contribute to telomere dynamics.     

High frequency excision of Ty elements during transformation of yeast.

Yeast (Saccharomyces cerevisiae) transposons (Ty elements) are excised from up to 20% of supercoiled plasmids during transformation of yeast cells. The excision occurs by homologous recombination across the direct terminal repeats (deltas) of the Ty element, leaving behind a single delta in the transforming plasmid. Only the initial transforming plasmid is susceptible to excision, and no high frequency excision is observed in plasmids that have become established in transformed cells or in plasmids that are resident in cells undergoing transformation. High frequency excision from plasmids during yeast transformation is not specific for Ty elements and can be observed with other segments of plasmid DNA bounded by direct repeats. The frequency of Ty excision from supercoiled plasmids is greatly reduced when the host yeast cells contain the rad52 mutation, a defect in double-strand DNA repair. When linear or ligated-linear plasmid DNAs containing a Ty element are used for transformation, few or no excision plasmids are found among the transformant colonies. These results suggest that when a yeast cell is transformed with a supercoiled plasmid, the plasmid DNA is highly susceptible to homologous recombination for a short period of time.     

Heterogeneous Functional Ty1 Elements Are Abundant in the Saccharomyces Cerevisiae Genome

Despite the abundance of Ty1 RNA in Saccharomyces cerevisiae, Ty1 retrotransposition is a rare event. To determine whether transpositional dormancy is the result of defective Ty1 elements, functional and defective alleles of the retrotransposon in the yeast genome were quantitated. Genomic Ty1 elements were isolated by gap repair-mediated recombination of pGTy1-H3(Δ475-3944)HIS3, a multicopy plasmid containing a GAL1/Ty1-H3 fusion element lacking most of the gag domain (TYA) and the protease (PR) and integrase (IN) domains. Of 39 independent gap repaired pGTyHIS3 elements isolated, 29 (74%) transposed at high levels following galactose induction. The presence of restriction site polymorphisms within the gap repaired region of the 29 functional pGTyHIS3 elements indicated that they were derived from at least eight different genomic Ty1 elements and one Ty2 element. Of the 10 defective pGTyHIS3 elements, one was a partial gap repair event while the other nine were derived from at least six different genomic Ty1 elements. These results suggest that most genomic Ty1 elements encode functional TYA, PR and IN proteins. To understand how functional Ty1 elements are regulated, we tested the hypothesis that a TYB protein associates preferentially in cis with the RNA template that encodes it, thereby promoting transposition of its own element. A genomic Ty1 mhis3AI element containing either an in-frame insertion in PR or a deletion in TYB transposed at the same rate as a wild-type Ty1mhis3AI allele, indicating that TYB proteins act efficiently in trans. This result suggests in principle that defective genomic Ty1 elements could encode trans-acting repressors of transposition; however, expression of only one of the nine defective pGTy1 isolates had a negative effect on genomic Ty1 mhis3AI element transposition in trans, and this effect was modest. Therefore, the few defective Ty1 elements in the genome are not responsible for transpositional dormancy.     

Allelic and Ectopic Recombination between Ty Elements in Yeast

Allelic and nonallelic (ectopic) recombination events were analyzed in a set of isogenic strains that carry marked Ty elements. We found that allelic recombination between Ty elements occurred at normal frequencies both in meiosis and mitosis. The marked Ty elements were involved in a large variety of different types of ectopic recombination and this variety was greater in mitosis than in meiosis. Allelic and ectopic recombination events occurred at similar frequencies in mitosis, but allelic recombination predominated in meiosis. Some of the types of ectopic mitotic recombination indicated the common occurrence of concerted recombination events. The length of homology represented by a delta element (330 bp) seemed to be sufficient for some types of mitotic and meiotic recombination.     

Recombination of Ty Elements in Yeast Can Be Induced by a Double-Strand Break

The Ty retrotransposons are the main family of dispersed repeated sequences in the yeast Saccharomyces cerevisiae. These elements are flanked by a pair of long terminal direct repeats (LTRs). Previous experiments have shown that Ty elements recombine at low frequencies, despite the fact that they are present in 30 copies per genome. This frequency is not highly increased by treatments that cause DNA damage, such as UV irradiation. In this study, we show that it is possible to increase the recombination level of a genetically marked Ty by creating a double-strand break in it. This break is repaired by two competing mechanisms: one of them leaves a single LTR in place of the Ty, and the other is a gene conversion event in which the marked Ty is replaced by an ectopically located one. In a strain in which the marked Ty has only one LTR, the double-strand break is repaired by conversion. We have also measured the efficiency of repair and monitored the progression of the cells through the cell-cycle. We found that in the presence of a double-strand break in the marked Ty, a proportion of the cells is unable to resume growth.     

High Frequency Cdna Recombination of the Saccharomyces Retrotransposon Ty5: The Ltr Mediates Formation of Tandem Elements

Retroelement cDNA can integrate into the genome using the element-encoded integrase, or it can recombine with preexisting elements using the recombination system of the host. Recombination is a particularly important pathway for the yeast retrotransposon Ty5 and accounts for ~30% of the putative transposition events when a homologous substrate is carried on a plasmid and ~7% when the substrate is located at the chromosomal URA3 locus. Characterization of recombinants revealed that they are either simple replacements of the marker gene or tandem elements. Using an assay system in which the donor element and recombination substrates are separated, we found that the long terminal repeats (LTRs) are critical for tandem element formation. LTR-containing substrates generate tandem elements at frequencies more than 10-fold higher than similarly sized internal Ty5 sequences. Internal sequences, however, facilitate tandem element formation when associated with an LTR, and there is a linear relationship between frequencies of tandem element formation and the length of LTR-containing substrates. We propose that recombination is initiated between the LTRs of the cDNA and substrate and that internal sequences promote tandem element formation by facilitating sequence alignment. Because of its location in subtelomeric regions, recombinational amplification of Ty5 may contribute to the organization of chromosome ends.     

Increase in Ty1 cDNA recombination in yeast sir4 mutant strains at high temperature

Transposition of the Ty1 element of the yeast Saccharomyces cerevisiae is temperature sensitive. We have identified a null allele of the silent information regulator gene SIR4 as a host mutant that allows for transposition at high temperature. We show that the apparent increase in transposition activity in sir4 mutant strains at high temperature is dependent on the RAD52 gene and is thus likely resulting from an increase in Ty1 cDNA recombination, rather than in IN-mediated integration. General cellular recombination is not increased at high temperature, suggesting that the increase in recombination at high temperature in sir4 mutants is specific for Ty1 cDNA. Additionally, this high-temperature Ty1 recombination was found to be dependent on functional Sir2p and Sir3p. We speculate that the increase in recombination seen in sir4 mutants at high temperature may be due to changes in chromatin structure or Ty1 interactions with chromosomal structures resulting in higher recombination rates.