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In Saccharomyces cerevisiae, heterochromatin structures required for transcriptional silencing of the HML and HMR loci are duplicated in coordination with passing DNA replication forks. Despite major reorganization of chromatin structure, the heterochromatic, transcriptionally silent states of HML and HMR are successfully maintained throughout S-phase. Mutations of specific components of the replisome diminish the capacity to maintain silencing of HML and HMR through replication. Similarly, mutations in histone chaperones involved in replication-coupled nucleosome assembly reduce gene silencing. Bridging these observations, we determined that the proliferating cell nuclear antigen (PCNA) unloading activity of Elg1 was important for coordinating DNA replication forks with the process of replication-coupled nucleosome assembly to maintain silencing of HML and HMR through S-phase. Collectively, these data identified a mechanism by which chromatin reassembly is coordinated with DNA replication to maintain silencing through S-phase.

Glycans form the primary nutritional source for microbes in the human gut, and understanding their metabolism is a critical yet understudied aspect of microbiome research. Here, we present a novel computational pipeline for modeling glycan degradation (GlyDeR) which predicts the glycan degradation potency of 10,000 reference glycans based on either genomic or metagenomic data. We first validated GlyDeR by comparing degradation profiles for genomes in the Human Microbiome Project against KEGG reaction annotations. Next, we applied GlyDeR to the analysis of human and mammalian gut microbial communities, which revealed that the glycan degradation potential of a community is strongly linked to host diet and can be used to predict diet with higher accuracy than sequence data alone. Finally, we show that a microbe's glycan degradation potential is significantly correlated (R = 0.46) with its abundance, with even higher correlations for potential pathogens such as the class Clostridia (R = 0.76). GlyDeR therefore represents an important tool for advancing our understanding of bacterial metabolism in the gut and for the future development of more effective prebiotics for microbial community manipulation. The increased availability of high-throughput sequencing data has positioned the gut microbiota as a major new focal point for biomedical research. However, despite the expenditure of huge efforts and resources, sequencing-based analysis of the microbiome has uncovered mostly associative relationships between human health and diet, rather than a causal, mechanistic one. In order to utilize the full potential of systems biology approaches, one must first characterize the metabolic requirements of gut bacteria, specifically, the degradation of glycans, which are their primary nutritional source. We developed a computational framework called GlyDeR for integrating expert knowledge along with high-throughput data to uncover important new relationships within glycan metabolism. GlyDeR analyzes particular bacterial (meta)genomes and predicts the potency by which they degrade a variety of different glycans. Based on GlyDeR, we found a clear connection between microbial glycan degradation and human diet, and we suggest a method for the rational design of novel prebiotics.

Abstract Background During evolution selection forces such as changing environments shape the architecture of genomes. The distribution of genes along chromosomes and the length of intragenic regions are basic genomic features known to play a major role in the regulation of gene transcription and translation. Results In this work we perform the first large scale analysis of the length distribution of untranslated regions (promoters, 5' and 3' untranslated regions, terminators) in the genome of the yeast Saccharomyces cerevisiae. Our analysis shows that the length of each open reading frame (ORF) and that of its associated regulatory and untranslated regions significantly correlate with each other. Moreover, significant correlations with other features related to gene expression and evolution (number of regulating transcription factors, mRNA and protein abundance, evolutionary rate, etc) were observed. Furthermore, the function of genes seems to have an important role in the evolution of these lengths. Notably, genes that are related to RNA metabolism tend to have shorter untranslated regions and thus tend to be closer to their neighbouring genes while genes coding for cell wall proteins tend to be isolated in the genome. Conclusion These results indicate that genome architecture has a significant role in regulating gene expression, and in shaping the characteristics and functionality of proteins.

Abstract Background Recent studies in a growing number of organisms have yielded accumulating evidence that a significant portion of the non-coding region in the genome is transcribed. We address this issue in the yeast Saccharomyces cerevisiae. Results Taking into account the absence of a significantly large yeast EST database, we use microarray expression data collected for genomic regions erroneously believed to be coding to study the expression pattern of non-coding regions in the Saccharomyces cerevisiae genome. We find that at least 164 out of 589 (28%) such regions are expressed under specific biological conditions. In particular, looking at the probes that are located opposing other known genes at the same genomic locus, we find that 88 out of 341 (26%) of these genes support antisense transcription. The expression patterns of these antisense genes are positively correlated. We validate these results using RT-PCR on a sample of 6 non-coding transcripts. Conclusion 1. The yeast genome is transcribed on a scale larger than previously assumed. 2. Correlated transcription of antisense genes is abundant in the yeast genome. 3. Antisense genes in yeast are non-coding.

Ribosome queuing is a fundamental phenomenon suggested to be related to topics such as genome evolution, synthetic biology, gene expression regulation, intracellular biophysics, and more. However, this phenomenon hasn't been quantified yet at a genomic level. Nevertheless, methodologies for studying translation (e.g. ribosome footprints) are usually calibrated to capture only single ribosome protected footprints (mRPFs) and thus limited in their ability to detect ribosome queuing. On the other hand, most of the models in the field assume and analyze a certain level of queuing. Here we present an experimental-computational approach for studying ribosome queuing based on sequencing of RNA footprints extracted from pairs of ribosomes (dRPFs) using a modified ribosome profiling protocol. We combine our approach with traditional ribosome profiling to generate a detailed profile of ribosome traffic. The data are analyzed using computational models of translation dynamics. The approach was implemented on the Saccharomyces cerevisiae transcriptome. Our data shows that ribosome queuing is more frequent than previously thought: the measured ratio of ribosomes within dRPFs to mRPFs is 0.2–0.35, suggesting that at least one to five translating ribosomes is in a traffic jam; these queued ribosomes cannot be captured by traditional methods. We found that specific regions are enriched with queued ribosomes, such as the 5’-end of ORFs, and regions upstream to mRPF peaks, among others. While queuing is related to higher density of ribosomes on the transcript (characteristic of highly translated genes), we report cases where traffic jams are relatively more severe in lowly expressed genes and possibly even selected for. In addition, our analysis demonstrates that higher adaptation of the coding region to the intracellular tRNA levels and longer genes are associated with lower queuing levels. Our analysis also suggests that the Saccharomyces cerevisiae transcriptome undergoes selection for eliminating traffic jams. Thus, our proposed approach is an essential tool for high resolution analysis of ribosome traffic during mRNA translation and understanding its evolution. [ABSTRACT FROM AUTHOR]

Protein-protein interactions (PPIs) govern basic cellular processes through signal transduction and complex formation. The diversity of those processes gives rise to a remarkable diversity of interactions types, ranging from transient phosphorylation interactions to stable covalent bonding. Despite our increasing knowledge on PPIs in humans and other species, their types remain relatively unexplored and few annotations of types exist in public databases. Here, we propose the first method for systematic prediction of PPI type based solely on the techniques by which the interaction was detected. We show that different detection methods are better suited for detecting specific types. We apply our method to ten interaction types on a large scale human PPI dataset. We evaluate the performance of the method using both internal cross validation and external data sources. In cross validation, we obtain an area under receiver operating characteristic (ROC) curve ranging from 0.65 to 0.97 with an average of 0.84 across the predicted types. Comparing the predicted interaction types to external data sources, we obtained significant agreements for phosphorylation and ubiquitination interactions, with hypergeometric p-value = 2.3e−54 and 5.6e−28 respectively. We examine the biological relevance of our predictions using known signaling pathways and chart the abundance of interaction types in cell processes. Finally, we investigate the cross-relations between different interaction types within the network and characterize the discovered patterns, or motifs. We expect the resulting annotated network to facilitate the reconstruction of process-specific subnetworks and assist in predicting protein function or interaction. [ABSTRACT FROM AUTHOR]

Replication-factor C (RFC) is a protein complex that loads the processivity clamp PCNA onto DNA. Elg1 is a conserved protein with homology to the largest subunit of RFC, but its function remained enigmatic. Here, we show that yeast Elg1 interacts physically and genetically with PCNA, in a manner that depends on PCNA modification, and exhibits preferential affinity for SUMOylated PCNA. This interaction is mediated by three small ubiquitin-like modifier (SUMO)-interacting motifs and a PCNA-interacting protein box close to the N-terminus of Elg1. These motifs are important for the ability of Elg1 to maintain genomic stability. SUMOylated PCNA is known to recruit the helicase Srs2, and in the absence of Elg1, Srs2 and SUMOylated PCNA accumulate on chromatin. Strains carrying mutations in both ELG1 and SRS2 exhibit a synthetic fitness defect that depends on PCNA modification. Our results underscore the importance of Elg1, Srs2 and SUMOylated PCNA in the maintenance of genomic stability. [ABSTRACT FROM AUTHOR]

To expand the known spectrum of genes that maintain genome stability, we screened a recently released collection of temperature sensitive (Ts) yeast mutants for a chromosome instability (CIN) phenotype. Proteasome subunit genes represented a major functional group, and subsequent analysis demonstrated an evolutionarily conserved role in CIN. Analysis of individual proteasome core and lid subunit mutations showed that the CIN phenotype at semi-permissive temperature is associated with failure of subunit localization to the nucleus. The resultant proteasome dysfunction affects chromosome stability by impairing the kinetics of double strand break (DSB) repair. We show that the DNA repair protein Mms22 is required for DSB repair, and recruited to chromatin in a ubiquitin-dependent manner as a result of DNA damage. Moreover, subsequent proteasome-mediated degradation of Mms22 is necessary and sufficient for cell cycle progression through the G2/M arrest induced by DNA damage. Our results demonstrate for the first time that a double strand break repair protein is a proteasome target, and thus link nuclear proteasomal activity and DSB repair. [ABSTRACT FROM AUTHOR]

Decades of research into homologous recombination have unravelled many of the details concerning the transfer of information between two homologous sequences. By contrast, the processes by which the interacting molecules initially colocalize are largely unknown. How can two homologous needles find each other in the genomic haystack? Is homologous pairing the result of a damage-induced homology search, or is it an enduring and general feature of the genomic architecture that facilitates homologous recombination whenever and wherever damage occurs? This Review presents the homologous-pairing enigma, delineates our current understanding of the process and offers guidelines for future research. [ABSTRACT FROM AUTHOR]

Genome stability is of primary importance for the survival and proper functioning of all organisms. Double-strand breaks (DSBs) arise spontaneously during growth, or can be created by external insults. Repair of DSBs by homologous recombination provides an efficient and fruitful pathway to restore chromosomal integrity. Exciting new work in yeast has lately provided insights into this complex process. Many of the proteins involved in recombination have been isolated and the details of the repair mechanism are now being unraveled at the molecular level. In this review, we focus on recent studies which dissect the recombinational repair of a single broken chromosome. After DSB formation, a decision is made regarding the mechanism of repair (recombination or non-homologous end-joining). This decision is under genetic control. Once committed to the recombination pathway, the broken chromosomal ends are resected by a still unclear mechanism in which the DNA damage checkpoint protein Rad24 participates. At this stage several proteins are recruited to the broken ends, including Rad51p, Rad52p, Rad55p, Rad57p, and possibly Rad54p. A genomic search for homology ensues, followed by strand invasion, promoted by the Rad51 filament with the participation of Rad55p, Rad57p and Rad54p. DNA synthesis then takes place, restoring the resected ends. Crossing-over formation depends on the length of the homologous recombining sequences, and is usually counteracted by the activity of the mismatch repair system. Given the conservation of the repair mechanisms and genes throughout evolution, these studies have profound implications for other eukaryotic organisms. [Copyright &y& Elsevier]

Homologous recombination plays a central role during meiosis, ensuring the proper segregation of homologous chromosomes during the first meiotic division. In addition, meiotic recombination generates genetic variability upon which natural selection can act. The frequency of recombination is not evenly distributed throughout the genome: regions of high (hotspots) and low (coldspots) recombination can be found. Meiotic hotspots exhibit high levels of double-strand break formation and these breaks coincide with the upstream regions of genes. In many cases, binding of transcription factors has been shown to be required for hotspot activity. We review the current knowledge on the mechanisms that determine hotspot activity and propose a modified model to account for recent observations which show that recombination frequency at hotspots is sensitive to environmental conditions. [ABSTRACT FROM AUTHOR]

The article discusses mechanisms responsible for the observed robustness in yeast, including the existence of duplicate genes providing backups to each other, alternative metabolic and signaling pathways and an intrinsic flexibility that stems from the need to accommodate a variety of potential growth environments. By systematically generating double knockouts of non-essential genes and assessing their fitness, it is possible to identify both positive and negative genetic interactions.

Recombination between repeated DNA sequences can have drastic consequences on the integrity of the genome. Repeated sequences are abundant in most eukaryotes, yet the mechanism that prevents recombination between them is currently unknown. Ty elements, the main family of dispersed repeats in Saccharomyces cerevisiae, exhibit low levels of exchange. Other regions in the genome have relatively high rates of meiotic recombination (hotspots). We show that a Ty element adjacent to the HIS4 recombination hotspot substantially reduces its activity, eliminating local DSB formation. We demonstrate that the Ty has a closed (nuclease-insensitive) chromatin configuration that is also imposed on the flanking DNA sequences. The compact chromatin structure is determined by sequences at the N terminus of the Ty. Increased binding of the Rap1 protein to the hotspot restores both open chromatin conformation and DSB formation. The chromatin configuration of Ty elements precludes initiation of recombination, thus preventing potentially lethal exchanges between repeated sequences. [Copyright &y& Elsevier]