Saccharomyces Mikatae Research Articles

INTERSPECIFIC HYBRID BETWEEN Saccharomyces mikatae AND Saccharomyces cerevisiae AS AN ALTERNATIVE STRAIN FOR BREAD MAKING

Hybrids of the homothallic diploid Saccharomyces mikatae and heterothallic haploid Saccharomyces cerevisiae isolated from local fruits in Hokkaido and a baking strain, respectively, were constructed by the spore to cell mating method. Interspecific hybridization of the selected strain was confirmed by polymerase chain reaction using species-specific primers, fermentation profiles, and spore formation. The leavening ability of the dough and enzyme activities of the hybrid exhibited intermediate traits of their parental strains. A baking test by the sponge-dough method with a standard formulation of white bread showed that breads produced using the hybrid differed in aroma and taste from those made using a conventional baking strain. Thus, the hybrid may be used to produce baked goods with diverse quality, which may be more acceptable to specific consumers.

Alternative Saccharomyces interspecies hybrid combinations and their potential for low-temperature wort fermentation.

The lager yeast hybrid (Saccharomyces cerevisiae × Saccharomyces eubayanus) possesses two key characteristics that are essential for lager brewing: efficient sugar utilization and cold tolerance. Here we explore the possibility that the lager yeast phenotype can be recreated by hybridizing S. cerevisiae ale yeast with a number of cold‐tolerant Saccharomyces species including Saccharomyces arboricola, Saccharomyces eubayanus, Saccharomyces mikatae and Saccharomyces uvarum. Interspecies hybrids performed better than parental strains in lager brewing conditions (12°C and 12°P wort), with the S. mikatae hybrid performing as well as the S. eubayanus hybrid. Where the S. cerevisiae parent was capable of utilizing maltotriose, this trait was inherited by the hybrids. A greater production of higher alcohols and esters by the hybrids resulted in the production of more aromatic beers relative to the parents. Strong fermentation performance relative to the parents was dependent on ploidy, with polyploid hybrids (3n, 4n) performing better than diploid hybrids. All hybrids produced 4‐vinyl guaiacol, a smoke/clove aroma generally considered an off flavour in lager beer. This characteristic could however be eliminated by isolating spore clones from a fertile hybrid of S. cerevisiae and S. mikatae. The results suggest that S. eubayanus is dispensable when constructing yeast hybrids that express the typical lager yeast phenotype. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.

Mitochondrial genome evolution in the Saccharomyces sensu stricto complex.

Exploring the evolutionary patterns of mitochondrial genomes is important for our understanding of the Saccharomyces sensu stricto (SSS) group, which is a model system for genomic evolution and ecological analysis. In this study, we first obtained the complete mitochondrial sequences of two important species, Saccharomyces mikatae and Saccharomyces kudriavzevii. We then compared the mitochondrial genomes in the SSS group with those of close relatives, and found that the non-coding regions evolved rapidly, including dramatic expansion of intergenic regions, fast evolution of introns and almost 20-fold higher rearrangement rates than those of the nuclear genomes. However, the coding regions, and especially the protein-coding genes, are more conserved than those in the nuclear genomes of the SSS group. The different evolutionary patterns of coding and non-coding regions in the mitochondrial and nuclear genomes may be related to the origin of the aerobic fermentation lifestyle in this group. Our analysis thus provides novel insights into the evolution of mitochondrial genomes.

Saccharomyces jurei sp. nov., isolation and genetic identification of a novel yeast species from Quercus robur.

Two strains, D5088T and D5095, representing a novel yeast species belonging to the genus Saccharomyces were isolated from oak tree bark and surrounding soil located at an altitude of 1000 m above sea level in Saint Auban, France. Sequence analyses of the internal transcribed spacer (ITS) region and 26S rRNA D1/D2 domains indicated that the two strains were most closely related to Saccharomyces mikatae and Saccharomyces paradoxus. Genetic hybridization analyses showed that both strains are reproductively isolated from all other Saccharomyces species and, therefore, represent a distinct biological species. The species name Saccharomyces jurei sp. nov. is proposed to accommodate these two strains, with D5088T (=CBS 14759T=NCYC 3947T) designated as the type strain.

Hybridization and adaptive evolution of diverse Saccharomyces species for cellulosic biofuel production

BackgroundLignocellulosic biomass is a common resource across the globe, and its fermentation offers a promising option for generating renewable liquid transportation fuels. The deconstruction of lignocellulosic biomass releases sugars that can be fermented by microbes, but these processes also produce fermentation inhibitors, such as aromatic acids and aldehydes. Several research projects have investigated lignocellulosic biomass fermentation by the baker’s yeast Saccharomyces cerevisiae. Most projects have taken synthetic biological approaches or have explored naturally occurring diversity in S. cerevisiae to enhance stress tolerance, xylose consumption, or ethanol production. Despite these efforts, improved strains with new properties are needed. In other industrial processes, such as wine and beer fermentation, interspecies hybrids have combined important traits from multiple species, suggesting that interspecies hybridization may also offer potential for biofuel research.ResultsTo investigate the efficacy of this approach for traits relevant to lignocellulosic biofuel production, we generated synthetic hybrids by crossing engineered xylose-fermenting strains of S. cerevisiae with wild strains from various Saccharomyces species. These interspecies hybrids retained important parental traits, such as xylose consumption and stress tolerance, while displaying intermediate kinetic parameters and, in some cases, heterosis (hybrid vigor). Next, we exposed them to adaptive evolution in ammonia fiber expansion-pretreated corn stover hydrolysate and recovered strains with improved fermentative traits. Genome sequencing showed that the genomes of these evolved synthetic hybrids underwent rearrangements, duplications, and deletions. To determine whether the genus Saccharomyces contains additional untapped potential, we screened a genetically diverse collection of more than 500 wild, non-engineered Saccharomyces isolates and uncovered a wide range of capabilities for traits relevant to cellulosic biofuel production. Notably, Saccharomyces mikatae strains have high innate tolerance to hydrolysate toxins, while some Saccharomyces species have a robust native capacity to consume xylose.ConclusionsThis research demonstrates that hybridization is a viable method to combine industrially relevant traits from diverse yeast species and that members of the genus Saccharomyces beyond S. cerevisiae may offer advantageous genes and traits of interest to the lignocellulosic biofuel industry.

Structure of the SLC4 transporter Bor1p in an inward-facing conformation.

Bor1p is a secondary transporter in yeast that is responsible for boron transport. Bor1p belongs to the SLC4 family which controls bicarbonate exchange and pH regulation in animals as well as borate uptake in plants. The SLC4 family is more distantly related to members of the Amino acid-Polyamine-organoCation (APC) superfamily, which includes well studied transporters such as LeuT, Mhp1, AdiC, vSGLT, UraA, SLC26Dg. Their mechanism generally involves relative movements of two domains: a core domain that binds substrate and a gate domain that in many cases mediates dimerization. To shed light on conformational changes governing transport by the SLC4 family, we grew helical membrane crystals of Bor1p from Saccharomyces mikatae and determined a structure at ∼6 Å resolution using cryo-electron microscopy. To evaluate the conformation of Bor1p in these crystals, a homology model was built based on the related anion exchanger from red blood cells (AE1). This homology model was fitted to the cryo-EM density map using the Molecular Dynamics (MD) Flexible Fitting method and then relaxed by all-atom MD simulation in explicit solvent and membrane. Mapping of water accessibility indicates that the resulting structure represents an inward-facing conformation. Comparisons of the resulting Bor1p model with the X-ray structure of AE1 in an outward-facing conformation, together with MD simulations of inward-facing and outward-facing Bor1p models, suggest rigid body movements of the core domain relative to the gate domain. These movements are consistent with the rocking-bundle transport mechanism described for other members of the APC superfamily.

Structure of the Borate Transporter Bor1p by cryo-EM

Boron is an essential micronutrient for plants and animals. Transport of boron regulates uptake and protects against high levels of boron in plants and yeast. The yeast boron transporter Bor1p belongs to the SLC4 Anion Exchanger family of transporters that also includes the human chloride/bicarbonate exchanger. We have overexpressed and purified the Bor1p orthologue from Saccharomyces mikatae using Saccharomyces cerevisiae as a host. Helical crystals were produced by reconstituting the transporter together with cardiolipin and electron micrographs of frozen-hydrated tubes were recorded with a field emission gun at 200 kV using a direct electron detector. Crystals adopted two different helical symmetries from which two independent reconstructions were produced from 75 particles at a resolution of ∼7 A using a Fourier-Bessel approach. In order to interpret the dimeric densities revealed by the reconstructions, we built a homology model for Bor1p based on the crystal structure of the uracil transporter (UraA), which is the closest relative to Anion Exchangers within the APC superfamily of transporters. Initial homology models were produced from 10 alternative alignments of these distantly related proteins. The alignments were refined by mapping sequence conservation onto these 3D structures. The best model was then fitted to the cryo-EM map by adjusting the position of individual helices and the resulting structure was equilibrated in a lipid bilayer for 200 ns using molecular dynamics. The resulting structure suggests conformational changes relative to UraA in which helices at the dimer interface are tilted relative to the transporter domain, thus providing access to the substrate binding site from the extracellular side of the membrane. Comparison of our Bor1p structure with that from UraA are likely to reflect the structural changes that accompany the alternating access mechanism employed by this family of transporters.

Yeast Interspecies Comparative Proteomics Reveals Divergence in Expression Profiles and Provides Insights into Proteome Resource Allocation and Evolutionary Roles of Gene Duplication

Omics analysis is a versatile approach for understanding the conservation and diversity of molecular systems across multiple taxa. In this study, we compared the proteome expression profiles of four yeast species (Saccharomyces cerevisiae, Saccharomyces mikatae, Kluyveromyces waltii, and Kluyveromyces lactis) grown on glucose- or glycerol-containing media. Conserved expression changes across all species were observed only for a small proportion of all proteins differentially expressed between the two growth conditions. Two Kluyveromyces species, both of which exhibited a high growth rate on glycerol, a nonfermentative carbon source, showed distinct species-specific expression profiles. In K. waltii grown on glycerol, proteins involved in the glyoxylate cycle and gluconeogenesis were expressed in high abundance. In K. lactis grown on glycerol, the expression of glycolytic and ethanol metabolic enzymes was unexpectedly low, whereas proteins involved in cytoplasmic translation, including ribosomal proteins and elongation factors, were highly expressed. These marked differences in the types of predominantly expressed proteins suggest that K. lactis optimizes the balance of proteome resource allocation between metabolism and protein synthesis giving priority to cellular growth. In S. cerevisiae, about 450 duplicate gene pairs were retained after whole-genome duplication. Intriguingly, we found that in the case of duplicates with conserved sequences, the total abundance of proteins encoded by a duplicate pair in S. cerevisiae was similar to that of protein encoded by nonduplicated ortholog in Kluyveromyces yeast. Given the frequency of haploinsufficiency, this observation suggests that conserved duplicate genes, even though minor cases of retained duplicates, do not exhibit a dosage effect in yeast, except for ribosomal proteins. Thus, comparative proteomic analyses across multiple species may reveal not only species-specific characteristics of metabolic processes under nonoptimal culture conditions but also provide valuable insights into intriguing biological principles, including the balance of proteome resource allocation and the role of gene duplication in evolutionary history.

High gene conversion frequency between genes encoding 2-deoxyglucose-6-phosphate phosphatase in 3 Saccharomyces species

Gene conversions are nonreciprocal sequence exchanges between genes. They are relatively common in Saccharomyces cerevisiae, but few studies have investigated the evolutionary fate of gene conversions or their functional impacts. Here, we analyze the evolution and impact of gene conversions between the two genes encoding 2-deoxyglucose-6-phosphate phosphatase in S. cerevisiae, Saccharomyces paradoxus and Saccharomyces mikatae. Our results demonstrate that the last half of these genes are subject to gene conversions among these three species. The greater similarity and the greater percentage of GC nucleotides in the converted regions, as well as the absence of long regions of adjacent common converted sites, suggest that these gene conversions are frequent and occur independently in all three species. The high frequency of these conversions probably result from the fact that they have little impact on the protein sequences encoded by these genes.

Introducing a New Breed of Wine Yeast: Interspecific Hybridisation between a Commercial Saccharomyces cerevisiae Wine Yeast and Saccharomyces mikatae

Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment.

Novel algorithms for accurate DNA base-calling

The ability to decipher the genetic code of different species would lead to significant future scientific achievements in important areas, including medicine and agriculture. The importance of DNA sequencing necessitated a need for efficient automation of identification of base sequences from traces generated by existing sequencing machines, a process referred to as DNA base-calling. In this paper, a pattern recognition technique was adopted to minimize the inaccuracy in DNA base-calling. Two new frameworks using Artificial Neural Networks and Polynomial Classifiers are proposed to model electropherogram traces belonging to Homo sapiens, Saccharomyces mikatae and Drosophila melanogaster. De-correlation, de-convolution and normalization were implemented as part of the pre-processing stage employed to minimize data imperfections attributed to the nature of the chemical reactions involved in DNA sequencing. Discriminative features that characterize each chromatogram trace were subsequently extracted and subjected to the chosen classifiers to categorize the events to their respective base classes. The models are trained such that they are not restricted to a specific species or to a specific chemical procedure of sequencing. The base- calling accuracy achieved is compared with the exist- ing standards, PHRED (Phil’s Read Editor) and ABI (Applied Biosystems, version2.1.1) KB base-callers in terms of deletion, insertion and substitution errors. Experimental evidence indicates that the proposed models achieve a higher base-calling accuracy when compared to PHRED and a comparable performance when compared to ABI. The results obtained demon- strate the potential of the proposed models for efficient and accurate DNA base-calling.

A multiplex set of species-specific primers for rapid identification of members of the genus Saccharomyces

The Saccharomyces genus (previously Saccharomyces sensu stricto) formally comprises Saccharomyces arboricola, Saccharomyces bayanus, Saccharomyces cariocanus, Saccharomyces cerevisiae, Saccharomyces kudriavzevii, Saccharomyces mikatae, Saccharomyces paradoxus and Saccharomyces pastorianus. Species-specific primer pairs that produce a single band of known and different product size have been developed for each member of the clade with the exception of S. pastorianus, which is a polyphyletic allopolyploid hybrid only found in lager breweries, and for which signature sequences could not be reliably created. Saccharomyces cariocanus is now regarded as an American variant of S. paradoxus, and accordingly a single primer pair that recognizes both species was developed. A different orthologous and essential housekeeping gene was used to detect each species, potentially avoiding competition between PCR primers and overlap between amplicons. In multiplex format, two or more different species could be identified in a single reaction; double and triple hybrids could not always be correctly identified. Forty-two unidentified yeasts from sugar cane juice fermentations were correctly identified as S. cerevisiae. A colony PCR method was developed that is rapid, robust, inexpensive and capable of automation, requires no mycological expertise on the part of the user and is thus useful for large-scale preliminary screens.

Identifying Gene Clusters within Localized Regions in Multiple Genomes

An important strategy to study genome evolution is to investigate the clustering of orthologous genes among multiple genomes, in which the most popular approaches require that the distance between adjacent genes in a cluster be small. We investigate a different formulation based on constraining the overall size of a cluster and develop statistical significance estimates that allow direct comparison of clusters of different sizes. We first consider a restricted version which requires that orthologous genes are strictly ordered within each cluster and show that it can be solved in polynomial time. We then develop practical exact algorithms for the unrestricted problem that allows paralogous genes within a genome and clusters that may not appear in every genome while considering a general model in which a gene is allowed to appear in more than one orthologous group. We show that our algorithm can identify biologically relevant gene clusters on four bacterial genomes Bacillus subtilis, Streptococcus pyogenes, Streptococcus pneumoniae, and Clostridium acetobutylicum. We also show that our algorithm can identify significantly more functionally enriched gene clusters on four yeast genomes Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces mikatae, and Saccharomyces bayanus than previous algorithms. A software program (GCFinder) and a list of gene clusters found on the bacterial and the yeast genomes are available at http://faculty.cse.tamu.edu/shsze/gcfinder .

Genetic identification of new biological species Saccharomyces arboricolus Wang et Bai

Direct genetic testing for hybrid sterility unambiguously showed that the newly described yeast Saccharomyces arboricolus Wang et Bai is reproductively isolated from Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces cariocanus, Saccharomyces kudriavzevii, Saccharomyces mikatae and Saccharomyces paradoxus and, therefore, represents a new biological species of the genus Saccharomyces. Combined phylogenetic analysis of the rDNA repeat sequences (18S, 26S, ITS), nuclear ACT1 and mitochondrial ATP9 genes revealed that S. arboricolus, along with S. kudriavzevii and S. bayanus, is distantly related to the other four biological species.

A multispecies-based taxonomic microarray reveals interspecies hybridization and introgression inSaccharomyces cerevisiae

A multispecies-based taxonomic microarray targeting coding sequences of diverged orthologous genes in Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces bayanus, Saccharomyces kudriavzevii, Naumovia castellii, Lachancea kluyveri and Candida glabrata was designed to allow identification of isolates of these species and their interspecies hybrids. Analysis of isolates of several Saccharomyces species and interspecies hybrids demonstrated the ability of the microarray to differentiate these yeasts on the basis of their specific hybridization patterns. Subsequent analysis of 183 supposed S. cerevisiae isolates of various ecological and geographical backgrounds revealed one misclassified S. bayanus or Saccharomyces uvarum isolate and four aneuploid interspecies hybrids, one between S. cerevisiae and S. bayanus and three between S. cerevisiae and S. kudriavzevii. Furthermore, this microarray design allowed the detection of multiple introgressed S. paradoxus DNA fragments in the genomes of three different S. cerevisiae isolates. These results show the power of multispecies-based microarrays as taxonomic tools for the identification of species and interspecies hybrids, and their ability to provide a more detailed characterization of interspecies hybrids and recombinants.

The evolution of 5S ribosomal RNA genes linked to the rDNA units of fungal species

5S ribosomal RNA (rRNA) genes are known to move in and out of various tandemly repeated eukaryotic gene families during evolution. Here, we investigated the organization of 5S rRNA genes linked to the ribosomal DNA (rDNA) units in 147 fungal species using available sequence and genome databanks. Whereas some fungal species have no 5S rRNA genes in their rDNA units, others have one or two 5S rRNA copies linked on the same or the opposite strand. There were at least 13 independent changes during the evolution of fungal species. These include two 5S rRNA genes loss, five 5S rRNA genes inversions and six 5S rRNA genes insertions (including duplications). The lower frequency of 5S rRNA genes loss might be due to the fact that these events are more likely to affect fitness. The maximum time required for 5S rRNA gene organization to change between related species was estimated to be 7.5 millions years based on the sequences of the elongation factor alpha genes of Candida glabrata and Saccharomyces mikatae. This time is much longer than the homogenization time predicted from theoretical and experimental studies and likely reflects the lack of closely related species or strains in our data set.

Predicting essential genes in fungal genomes

Essential genes are required for an organism's viability, and the ability to identify these genes in pathogens is crucial to directed drug development. Predicting essential genes through computational methods is appealing because it circumvents expensive and difficult experimental screens. Most such prediction is based on homology mapping to experimentally verified essential genes in model organisms. We present here a different approach, one that relies exclusively on sequence features of a gene to estimate essentiality and offers a promising way to identify essential genes in unstudied or uncultured organisms. We identified 14 characteristic sequence features potentially associated with essentiality, such as localization signals, codon adaptation, GC content, and overall hydrophobicity. Using the well-characterized baker's yeast Saccharomyces cerevisiae, we employed a simple Bayesian framework to measure the correlation of each of these features with essentiality. We then employed the 14 features to learn the parameters of a machine learning classifier capable of predicting essential genes. We trained our classifier on known essential genes in S. cerevisiae and applied it to the closely related and relatively unstudied yeast Saccharomyces mikatae. We assessed predictive success in two ways: First, we compared all of our predictions with those generated by homology mapping between these two species. Second, we verified a subset of our predictions with eight in vivo knockouts in S. mikatae, and we present here the first experimentally confirmed essential genes in this species.

A surprisingly large RNase P RNA in Candida glabrata

We have found an extremely large ribonuclease P (RNase P) RNA (RPR1) in the human pathogen Candida glabrata and verified that this molecule is expressed and present in the active enzyme complex of this hemiascomycete yeast. A structural alignment of the C. glabrata sequence with 36 other hemiascomycete RNase P RNAs (abbreviated as P RNAs) allows us to characterize the types of insertions. In addition, 15 P RNA sequences were newly characterized by searching in the recently sequenced genomes Candida albicans, C. glabrata, Debaryomyces hansenii, Eremothecium gossypii, Kluyveromyces lactis, Kluyveromyces waltii, Naumovia castellii, Saccharomyces kudriavzevii, Saccharomyces mikatae, and Yarrowia lipolytica; and by PCR amplification for other Candida species (Candida guilliermondii, Candida krusei, Candida parapsilosis, Candida stellatoidea, and Candida tropicalis). The phylogenetic comparative analysis identifies a hemiascomycete secondary structure consensus that presents a conserved core in all species with variable insertions or deletions. The most significant variability is found in C. glabrata P RNA in which three insertions exceeding in total 700 nt are present in the Specificity domain. This P RNA is more than twice the length of any other homologous P RNAs known in the three domains of life and is eight times the size of the smallest. RNase P RNA, therefore, represents one of the most diversified noncoding RNAs in terms of size variation and structural diversity.

Engineering evolution to study speciation in yeasts.

The Saccharomyces 'sensu stricto' yeasts are a group of species that will mate with one another, but interspecific pairings produce sterile hybrids. A retrospective analysis of their genomes revealed that translocations between the chromosomes of these species do not correlate with the group's sequence-based phylogeny (that is, translocations do not drive the process of speciation). However, that analysis was unable to infer what contribution such rearrangements make to reproductive isolation between these organisms. Here, we report experiments that take an interventionist, rather than a retrospective approach to studying speciation, by reconfiguring the Saccharomyces cerevisiae genome so that it is collinear with that of Saccharomyces mikatae. We demonstrate that this imposed genomic collinearity allows the generation of interspecific hybrids that produce a large proportion of spores that are viable, but extensively aneuploid. We obtained similar results in crosses between wild-type S. cerevisiae and the naturally collinear species Saccharomyces paradoxus, but not with non-collinear crosses. This controlled comparison of the effect of chromosomal translocation on species barriers suggests a mechanism for the generation of redundancy in the S. cerevisiae genome.

Uncoupling of 3′-Phosphatase and 5′-Kinase Functions in Budding Yeast: CHARACTERIZATION OF SACCHAROMYCES CEREVISIAE DNA 3′-PHOSPHATASE (TPP1)

Polynucleotide kinase is a bifunctional enzyme containing both DNA 3'-phosphatase and 5'-kinase activities seemingly suited to the coupled repair of single-strand nicks in which the phosphate has remained with the 3'-base. We show that the yeast Saccharomyces cerevisiae is able to repair transformed dephosphorylated linear plasmids by non-homologous end joining with considerable efficiency independently of the end-processing polymerase Pol4p. Homology searches and biochemical assays did not reveal a 5'-kinase that would account for this repair, however. Instead, open reading frame YMR156C (here named TPP1) is shown to encode only a polynucleotide kinase-type 3'-phosphatase. Tpp1p bears extensive similarity to the ancient L-2-halo-acid dehalogenase and DDDD phosphohydrolase superfamilies, but is specific for double-stranded DNA. It is present at high levels in cell extracts in a functional form and so does not represent a pseudogene. Moreover, the phosphatase-only nature of this gene is shared by Saccharomyces mikatae YMR156C and Arabidopsis thaliana K15M2.3. Repair of 3'-phosphate and 5'-hydroxyl lesions is thus uncoupled in budding yeast as compared with metazoans. Repair of transformed dephosphorylated plasmids, and 5'-hydroxyl blocking lesions more generally, likely proceeds by a cycle of base removal and resynthesis.