Ectopic recombination between Ty elements in Saccharomyces cerevisiae is not induced by DNA damage.

Mitotic recombination is increased when cells are treated with a variety of physical and chemical agents that cause damage to their DNA. We show here, using Saccharomyces cerevisiae strains that carry marked Ty elements, that recombination between members of this family of retrotransposons is not increased by UV irradiation or by treatment with the radiomimetic drug methyl methanesulfonate. Both ectopic recombination and mutation events were elevated by these agents for non-Ty sequences in the same strain. We discuss possible mechanisms that can prevent the induction of recombination between Ty elements.     

Proteolytic processing of Ty3 proteins is required for transposition.

Ty3 is a retroviruslike element found in Saccharomyces cerevisiae. It encodes GAG3 and GAG3-POL3 polyproteins which are processed into mature proteins found in the Ty3 viruslike particle. In this study, the region encoding a protease that is homologous to retroviral aspartyl proteases was identified and shown to be required for production of mature Ty3 proteins and transposition. The Ty3 protease has the Asp-Ser-Gly consensus sequence found in copia, Ty1, and Rous sarcoma virus proteases, rather than the Asp-Thr-Gly found in most retroviral proteases. The Asp-Ser-Gly consensus is flanked by residues similar to those which flank the active sites of cellular aspartyl proteases. Mutations were made in the Ty3 active-site sequence to examine the role of the protease in Ty3 particle maturation and to test the functional significance of the Ser active-site variant in the consensus sequence. Mutation of the active-site Asp blocked processing of Gag3 and Gag3-Pol3 and allowed identification of a GAG3-POL3 polyprotein. This protein was turned over rapidly in cells expressing the mutant Ty3. Changing the active-site Ser to Thr caused only a modest reduction in the levels of certain Ty3 proteins. Five putative cleavage sites of this protease in Ty3 GAG3 and GAG3-POL3 polyproteins were defined by amino-terminal sequence analysis. The existence of an additional protein(s) of unknown function, encoded downstream of the protease-coding region, was deduced from the positions of these amino termini and the sizes of known Ty3 proteins. Although Ty3 protease cleavage sites do not correspond exactly to known retroviral protease cleavage sites, there are similarities. Residues P3 through P2' in the regions encompassing each of the five sites are uncharged, and no P1 position is occupied by an amino acid with a branched beta carbon.     

Genetic mapping of Ty elements in Saccharomyces cerevisiae.

We used transformation to insert a selectable marker at various sites in the Saccharomyces cerevisiae genome occupied by the transposable element Ty. The vector CV9 contains the LEU2+ gene and a portion of the repeated element Ty1-17. Transformation with this plasmid resulted in integration of the vector via a reciprocal exchange using homology at the LEU2 locus or at the various Ty elements that are dispersed throughout the S. cerevisiae genome. These transformants were used to map genetically sites of several Ty elements. The 24 transformants recovered at Ty sites define 19 distinct loci. Seven of these were placed on the genetic map. Two classes of Ty elements were identified in these experiments: a Ty1-17 class and Ty elements different from Ty1-17. Statistical analysis of the number of transformants at each class of Ty elements shows that there is preferential integration of the CV9 plasmid into the Ty1-17 class.     

Replication protein A is required for meiotic recombination in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, meiotic recombination is initiated by transient DNA double-stranded breaks (DSBs). These DSBs undergo a 5' --> 3' resection to produce 3' single-stranded DNA ends that serve to channel DSBs into the RAD52 recombinational repair pathway. In vitro studies strongly suggest that several proteins of this pathway--Rad51, Rad52, Rad54, Rad55, Rad57, and replication protein A (RPA)--play a role in the strand exchange reaction. Here, we report a study of the meiotic phenotypes conferred by two missense mutations affecting the largest subunit of RPA, which are localized in the protein interaction domain (rfa1-t11) and in the DNA-binding domain (rfa1-t48). We find that both mutant diploids exhibit reduced sporulation efficiency, very poor spore viability, and a 10- to 100-fold decrease in meiotic recombination. Physical analyses indicate that both mutants form normal levels of meiosis-specific DSBs and that the broken ends are processed into 3'-OH single-stranded tails, indicating that the RPA complex present in these rfa1 mutants is functional in the initial steps of meiotic recombination. However, the 5' ends of the broken fragments undergo extensive resection, similar to what is observed in rad51, rad52, rad55, and rad57 mutants, indicating that these RPA mutants are defective in the repair of the Spo11-dependent DSBs that initiate homologous recombination during meiosis.     

The SNF3 gene is required for high-affinity glucose transport in Saccharomyces cerevisiae.

Glucose uptake mutants have not been previously obtained in Saccharomyces cerevisiae, possibly because there seem to be at least two transport systems, of low and high affinities. We showed that snf3 (sucrose nonfermenting) mutants did not express high-affinity glucose uptake. Furthermore, their growth was completely impaired on low concentrations of glucose in the presence of antimycin A (which blocks respiration). Several genes which complemented the original snf3 gene were obtained on multicopy plasmids. Some of them, as well as plasmid-carried SNF3 itself, conferred a substantial increase in high-affinity glucose uptake in both snf3 and wild-type hosts. The effects of glucose on the expression of such a plasmid-determined high-affinity uptake resembled those in the wild type. Other genes complementing snf3 seemed to cause an increase in low-affinity glucose uptake. We suggest that SNF3 may function specifically in high-affinity glucose uptake, which is needed under some conditions of growth on low glucose concentrations. SNF3 itself or the other complementing genes may specify components of the glucose uptake system.     

The ARS consensus sequence is required for chromosomal origin function in Saccharomyces cerevisiae.

Replication origins have been mapped to positions that coincide, within experimental error (several hundred base pairs), with ARS elements. To determine whether the DNA sequences required for ARS function on plasmids are required for chromosomal origin function, the chromosomal copy of ARS306 was deleted and the chromosomal copy of ARS307 was replaced with mutant derivatives of ARS307 containing single point mutations in domain A within the ARS core consensus sequence. The chromosomal origin function of these derivatives was assayed by two-dimensional agarose gel electrophoresis. Deletion of ARS306 deleted the associated replication origin. The effects on chromosomal origin function of mutations in domain A paralleled their effects on ARS function, as measured by plasmid stability. These results demonstrate that chromosomal origin function is a property of the ARS element itself.     

hsk1+, a Schizosaccharomyces pombe gene related to Saccharomyces cerevisiae CDC7, is required for chromosomal replication.

Degenerate oligonucleotide-directed polymerase chain reaction was conducted to clone a possible Schizosaccharomyces pombe homologue [hsk1 for a putative homologue of CDC7 (seven) kinase 1] of Saccharomyces cerevisiae Cdc7 kinase. The cloned cDNA for hsk1+ contains an open reading frame consisting of 507 amino acids with predicted mol. wt of 58,370 that possesses overall amino acid identity of 46% (65% including similar residues) to CDC7. In addition to conserved domains for serine-threonine kinases, the predicted primary structure of Hsk1 contains three 'kinase insert' sequences characteristic to Cdc7 at the positions identical to those of Cdc7. Whereas the length and sequences of the kinase inserts are diverged between the two yeast species, 58% identity (76% including similar residues) is detected within the kinase conserved domains. The hsk1+ gene, which is present as a single copy on the S.pombe chromosome, contains two introns within the coding frame. Disruption of the hsk1+ gene by insertion of the ura4+ gene is lethal to growth. Analysis of the DNA content of germinating spores that contain hsk1 null alleles indicates that DNA replication is inhibited in the mutant. The morphology of these mutant spores after germination indicates abnormal nuclear division in some population of germinating spores, suggesting either that Hsk1 may be required for inhibition of mitosis until completion of S phase or that it may also be involved in proper execution of mitosis. Our results suggest that hsk1+ is a strong candidate for the functional fission yeast homologue of budding yeast CDC7 and that a mechanism through which initiation of chromosomal replication is regulated may be conserved between the two yeast species.     

Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae.

Transformation of Saccharomyces cerevisiae strains was examined by using the URA3 and TRP1 genes cloned into M13 vectors in the absence of sequences capable of promoting autonomous replication. These constructs transform S. cerevisiae cells to prototrophy by homologous recombination with the resident mutant gene. Single-stranded DNA was found to transform S. cerevisiae cells at efficiencies greater than that of double-stranded DNA. No conversion of single-stranded transforming DNA into duplex forms could be detected during the transformation process, and we conclude that single-stranded DNA may participate directly in recombination with chromosomal sequences. Transformation with single-stranded DNA gave rise to both gene conversion and reciprocal exchange events. Cotransformation with competing heterologous single-stranded DNA specifically inhibited transformation by single-stranded DNA, suggesting that one of the components in the transformation-recombination process has a preferential affinity for single-stranded DNA.     

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.     

The HXT2 gene of Saccharomyces cerevisiae is required for high-affinity glucose transport.

The HXT2 gene of the yeast Saccharomyces cerevisiae was identified on the basis of its ability to complement the defect in glucose transport of a snf3 mutant when present on the multicopy plasmid pSC2. Analysis of the DNA sequence of HXT2 revealed an open reading frame of 541 codons, capable of encoding a protein of Mr 59,840. The predicted protein displayed high sequence and structural homology to a large family of procaryotic and eucaryotic sugar transporters. These proteins have 12 highly hydrophobic regions that could form transmembrane domains; the spacing of these putative transmembrane domains is also highly conserved. Several amino acid motifs characteristic of this sugar transporter family are also present in the HXT2 protein. An hxt2 null mutant strain lacked a significant component of high-affinity glucose transport when under derepressing (low-glucose) conditions. However, the hxt2 null mutation did not incur a major growth defect on glucose-containing media. Genetic and biochemical analyses suggest that wild-type levels of high-affinity glucose transport require the products of both the HXT2 and SNF3 genes; these genes are not linked. Low-stringency Southern blot analysis revealed a number of other sequences that cross-hybridize with HXT2, suggesting that S. cerevisiae possesses a large family of sugar transporter genes.