Checkpoint phosphorylation sites on budding yeast Rif1 protect nascent DNA from degradation by Sgs1-Dna2.

In budding yeast the Rif1 protein is important for protecting nascent DNA at blocked replication forks, but the mechanism has been unclear. Here we show that budding yeast Rif1 must interact with Protein Phosphatase 1 to protect nascent DNA. In the absence of Rif1, removal of either Dna2 or Sgs1 prevents nascent DNA degradation, implying that Rif1 protects nascent DNA by targeting Protein Phosphatase 1 to oppose degradation by the Sgs1-Dna2 nuclease-helicase complex. This functional role for Rif1 is conserved from yeast to human cells. Yeast Rif1 was previously identified as a target of phosphorylation by the Tel1/Mec1 checkpoint kinases, but the importance of this phosphorylation has been unclear. We find that nascent DNA protection depends on a cluster of Tel1/Mec1 consensus phosphorylation sites in the Rif1 protein sequence, indicating that the intra-S phase checkpoint acts to protect nascent DNA through Rif1 phosphorylation. Our observations uncover the pathway by which budding yeast Rif1 stabilises newly synthesised DNA, highlighting the crucial role Rif1 plays in maintaining genome stability from lower eukaryotes to humans.

Evolutionary journey and characterisation of a novel pan-gene associated with beer strains of Saccharomyces cerevisiae.

The sequencing of over a thousand Saccharomyces cerevisiae genomes revealed a complex pangenome. Over one third of the discovered genes are not present in the S. cerevisiae core genome but instead are often restricted to a subset of yeast isolates and thus may be important for adaptation to specific environmental niches. We refer to these genes as "pan-genes," being part of the pangenome but not the core genome. Here, we describe the evolutionary journey and characterisation of a novel pan-gene, originally named hypothetical (HYPO) open-reading frame. Phylogenetic analysis reveals that HYPO has been predominantly retained in S. cerevisiae strains associated with brewing but has been repeatedly lost in most other fungal species during evolution. There is also evidence that HYPO was horizontally transferred at least once, from S. cerevisiae to Saccharomyces paradoxus. The phylogenetic analysis of HYPO exemplifies the complexity and intricacy of evolutionary trajectories of genes within the S. cerevisiae pangenome. To examine possible functions for Hypo, we overexpressed a HYPO-GFP fusion protein in both S. cerevisiae and Saccharomyces pastorianus. The protein localised to the plasma membrane where it accumulated initially in distinct foci. Time-lapse fluorescent imaging revealed that when cells are grown in wort, Hypo-gfp fluorescence spreads throughout the membrane during cell growth. The overexpression of Hypo-gfp in S. cerevisiae or S. pastorianus strains did not significantly alter cell growth in medium-containing glucose, maltose, maltotriose, or wort at different concentrations.

Budding yeast Rif1 binds to replication origins and protects DNA at blocked replication forks.

Despite its evolutionarily conserved function in controlling DNA replication, the chromosomal binding sites of the budding yeast Rif1 protein are not well understood. Here, we analyse genome-wide binding of budding yeast Rif1 by chromatin immunoprecipitation, during G1 phase and in S phase with replication progressing normally or blocked by hydroxyurea. Rif1 associates strongly with telomeres through interaction with Rap1. By comparing genomic binding of wild-type Rif1 and truncated Rif1 lacking the Rap1-interaction domain, we identify hundreds of Rap1-dependent and Rap1-independent chromosome interaction sites. Rif1 binds to centromeres, highly transcribed genes and replication origins in a Rap1-independent manner, associating with both early and late-initiating origins. Interestingly, Rif1 also binds around activated origins when replication progression is blocked by hydroxyurea, suggesting association with blocked forks. Using nascent DNA labelling and DNA combing techniques, we find that in cells treated with hydroxyurea, yeast Rif1 stabilises recently synthesised DNA Our results indicate that, in addition to controlling DNA replication initiation, budding yeast Rif1 plays an ongoing role after initiation and controls events at blocked replication forks.

The hybrid genomes of Saccharomyces pastorianus: A current perspective.

Saccharomyces pastorianus is a recently evolved interspecies hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus used in the production of lager-type beers and has a long-standing history with the brewing industry. At least two distinct types of lager yeasts (Groups I and II) have been identified based on chromosome content and structure. One important feature of the genomes of lager yeasts is the presence of a set of hybrid chromosomes that emerged as a result of homeologous recombination events between the parental chromosomes. The unique genetic composition of the hybrid genomes of S. pastorianus affords interesting opportunities for evolution, adaptation and survival of the hybrids. The co-expression of S. eubayanus, S. cerevisiae and hybrid gene alleles, together with gene dosage effects resulting from the presence of multiple copies of individual genes, creates a complex algorithm for gene expression, cellular biochemistry and physiology. The recent availability of genome sequences for three Group I and ten Group II lager yeast strains provides an opportunity to decipher this complex algorithm and understand how it impacts on the final fermentation product: flavoursome beer. Copyright © 2017 John Wiley & Sons, Ltd.

Recombination sites on hybrid chromosomes in Saccharomyces pastorianus share common sequence motifs and define a complex evolutionary relationship between group I and II lager yeasts.

Saccharomyces pastorianus, referred to as lager yeasts, are hybrids of S. cerevisiae and S. eubayanus. Isolates within the species are divided into two groups (I and II) based on chromosome structure and composition. Following the hybridisation, the parental chromosomes underwent homeologous recombination, generating a set of hybrid chromosomes unique to the species. Here, we assessed the recombination events in seven lager yeast genomes to more clearly define the evolutionary route of lager yeasts. Meta-analysis of the recombination epicentres, as well as a detailed analysis of recombination events at the MAT locus, reveals a more complex evolutionary relationship between the group I and II lager yeasts than previously considered and identifies several divergent routes of evolution leading to the current S. pastorianus strains. We show that recombination epicentres contain sequential runs of pyrimidines, often flanked by purines, on one strand of the DNA, and identify two common sequence motifs present in >80% of the recombination epicentres, indicating that a common mechanism might account for the recombination events. Taken together, the data support a sequential hybridisation model of evolution for the two types of lager yeasts and suggest that the genomes of this newly emerged species are highly dynamic and continually evolving.

Brewing up a storm: The genomes of lager yeasts and how they evolved.

Yeasts used in the production of lager beers belong to the species Saccharomyces pastorianus, an interspecies hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus. The hybridisation event happened approximately 500-600years ago and therefore S. pastorianus may be considered as a newly evolving species. The happenstance of the hybridisation event created a novel species, with unique genetic characteristics, ideal for the fermentation of sugars to produce flavoursome beer. Lager yeast strains retain the chromosomes of both parental species and also have sets of novel hybrid chromosomes that arose by recombination between the homeologous parental chromosomes. The lager yeasts are subdivided into two groups (I and II) based on the S. cerevisiae: S. eubayanus gene content and the types and numbers of hybrid chromosomes. Recently, whole genome sequences for several Group I and II lager yeasts and for many S. cerevisiae and S. eubayanus isolates have become available. Here we review the available genome data and discuss the likely origins of the parental species that gave rise to S. pastorianus. We review the compiled data on the composition of the lager yeast genomes and consider several evolutionary models to account for the emergence of the two distinct types of lager yeasts.

Loss of lager specific genes and subtelomeric regions define two different Saccharomyces cerevisiae lineages for Saccharomyces pastorianus Group I and II strains.

Lager yeasts, Saccharomyces pastorianus, are interspecies hybrids between S. cerevisiae and S. eubayanus and are classified into Group I and Group II clades. The genome of the Group II strain, Weihenstephan 34/70, contains eight so-called 'lager-specific' genes that are located in subtelomeric regions. We evaluated the origins of these genes through bioinformatic and PCR analyses of Saccharomyces genomes. We determined that four are of cerevisiae origin while four originate from S. eubayanus. The Group I yeasts contain all four S. eubayanus genes but individual strains contain only a subset of the cerevisiae genes. We identified S. cerevisiae strains that contain all four cerevisiae 'lager-specific' genes, and distinct patterns of loss of these genes in other strains. Analysis of the subtelomeric regions uncovered patterns of loss in different S. cerevisiae strains. We identify two classes of S. cerevisiae strains: ale yeasts (Foster O) and stout yeasts with patterns of 'lager-specific' genes and subtelomeric regions identical to Group I and II S. pastorianus yeasts, respectively. These findings lead us to propose that Group I and II S. pastorianus strains originate from separate hybridization events involving different S. cerevisiae lineages. Using the combined bioinformatic and PCR data, we describe a potential classification map for industrial yeasts.