Yeast as a model system to study metabolic impact of selenium compounds

Inorganic Se forms such as selenate or selenite (the two more abundant forms in nature) can be toxic in Saccharomyces cerevisiae cells, which constitute an adequate model to study such toxicity at the molecular level and the functions participating in protection against Se compounds. Those Se forms enter the yeast cell through other oxyanion transporters. Once inside the cell, inorganic Se forms may be converted into selenide through a reductive pathway that in physiological conditions involves reduced glutathione with its consequent oxidation into diglutathione and alteration of the cellular redox buffering capacity. Selenide can subsequently be converted by molecular oxygen into elemental Se, with production of superoxide anions and other reactive oxygen species. Overall, these events result in DNA damage and dose-dependent reversible or irreversible protein oxidation, although additional oxidation of other cellular macromolecules cannot be discarded. Stress-adaptation pathways are essential for efficient Se detoxification, while activation of DNA damage checkpoint and repair pathways protects against Se-mediated genotoxicity. We propose that yeast may be used to improve our knowledge on the impact of Se on metal homeostasis, the identification of Se-targets at the DNA and protein levels, and to gain more insights into the mechanism of Se-mediated apoptosis.España, Junta de Andalucía P11-CTS- 796

Non‐Canonical Replication Initiation: You’re Fired!

The division of prokaryotic and eukaryotic cells produces two cells that inherit a perfect copy of the genetic material originally derived from the mother cell. The initiation of canonical DNA replication must be coordinated to the cell cycle to ensure the accuracy of genome duplication. Controlled replication initiation depends on a complex interplay of cis‐acting DNA sequences, the so‐called origins of replication (ori), with trans‐acting factors involved in the onset of DNA synthesis. The interplay of cis‐acting elements and trans‐acting factors ensures that cells initiate replication at sequence‐specific sites only once, and in a timely order, to avoid chromosomal endoreplication. However, chromosome breakage and excessive RNA:DNA hybrid formation can cause breakinduced (BIR) or transcription‐initiated replication (TIR), respectively. These non‐canonical replication events are expected to affect eukaryotic genome function and maintenance, and could be important for genome evolution and disease development. In this review, we describe the difference between canonical and non‐canonical DNA replication, and focus on mechanistic differences and common features between BIR and TIR. Finally, we discuss open issues on the factors and molecular mechanisms involved in TIR.España, MINECO BFU2015-69183-

In vivo mapping of nucleosomes using psoralen-DNA crosslinking and primer extension

By the use of psoralen crosslinking and primer extension, a method was developed which allows the analysis of chromatin structure in vivo. Using a yeast minichromosome, >9 nucleosomes were mapped with a resolution of at least ±30 b

A new connection of mRNP biogenesis and export with transcription-coupled repair

Although DNA repair is faster in the transcribed strand of active genes, little is known about the possible contribution of mRNP biogenesis and export in transcription-coupled repair (TCR). Interestingly, mutants of THO, a transcription complex involved in maintenance of genome integrity, mRNP biogenesis and export, were recently found to be deficient in nucleotide excision repair. In this study we show by molecular DNA repair analysis, that Sub2-Yra1 and Thp1-Sac3, two main mRNA export complexes, are required for efficient TCR in yeast. Careful analysis revealed that THO mutants are also specifically affected in TCR. Ribozyme-mediated mRNA self-cleavage between two hot spots for UV damage showed that efficient TCR does not depend on the nascent mRNA, neither in wild-type nor in mutant cells. Along with severe UV damage-dependent loss in processivity, RNAPII was found binding to chromatin upon UV irradiation in THO mutants, suggesting that RNAPII remains stalled at DNA lesions. Furthermore, Def1, a factor responsible for the degradation of stalled RNAPII, appears essential for the viability of THO mutants subjected to DNA damage. Our results indicate that RNAPII is not proficient for TCR in mRNP biogenesis and export mutants, opening new perspectives on our knowledge of TCR in eukaryotic cells

Repair of UV-induced DNA lesions in natural Saccharomyces cerevisiae telomeres is moderated by Sir2 and Sir3, and inhibited by yKu–Sir4 interaction

Ultraviolet light (UV) causes DNA damage that is removed by nucleotide excision repair (NER). UVinduced DNA lesions must be recognized and repaired in nucleosomal DNA, higher order structures of chromatin and within different nuclear subcompartments. Telomeric DNA is made of short tandem repeats located at the ends of chromosomes and their maintenance is critical to prevent genome instability. In Saccharomyces cerevisiae the chromatin structure of natural telomeres is distinctive and contingent to telomeric DNA sequences. Namely, nucleosomes and Sir proteins form the heterochromatin like structure of X-type telomeres, whereas a more open conformation is present at Y’-type telomeres. It is proposed that there are no nucleosomes on the most distal telomeric repeat DNA, which is bound by a complex of proteins and folded into higher order structure. How these structures affect NER is poorly understood. Our data indicate that the X-type, but not the Y’-type, sub-telomeric chromatin modulates NER, a consequence of Sir protein-dependent nucleosome stability. The telomere terminal complex also prevents NER, however, this effect is largely dependent on the yKu–Sir4 interaction, but Sir2 and Sir3 independent

Role for RNA: DNA hybrids in origin-independent replication priming in a eukaryotic system

DNA replication initiates at defined replication origins along eukaryotic chromosomes, ensuring complete genome duplication within a single S-phase. A key feature of replication origins is their ability to control the onset of DNA synthesis mediated by DNA polymerase-α and its intrinsic RNA primase activity. Here, we describe a novel origin-independent replication process that is mediated by transcription. RNA polymerase I transcription constraints lead to persistent RNA:DNA hybrids (R-loops) that prime replication in the ribosomal DNA locus. Our results suggest that eukaryotic genomes have developed tools to prevent R-loop–mediated replication events that potentially contribute to copy number variation, particularly relevant to carcinogenesis

Manganese Stress Tolerance Depends on Yap1 and Stress-Activated MAP Kinases

Understanding which intracellular signaling pathways are activated by manganese stress is crucial to decipher how metal overload compromise cellular integrity. Here, we unveil a role for oxidative and cell wall stress signaling in the response to manganese stress in yeast. We find that the oxidative stress transcription factor Yap1 protects cells against manganese toxicity. Conversely, extracellular manganese addition causes a rapid decay in Yap1 protein levels. In addition, manganese stress activates the MAPKs Hog1 and Slt2 (Mpk1) and leads to an up-regulation of the Slt2 downstream transcription factor target Rlm1. Importantly, Yap1 and Slt2 are both required to protect cells from oxidative stress in mutants impaired in manganese detoxification. Under such circumstances, Slt2 activation is enhanced upon Yap1 depletion suggesting an interplay between different stress signaling nodes to optimize cellular stress responses and manganese tolerance.Universidad de Sevilla 2020/00001326Junta de Andalucía P20-RT-01220Ministerio de Ciencia e Innovación PID2019-105342GB-I0

Surface modulation of single-walled carbon nanotubes for selective bacterial cell agglutination

Background: Bacterial resistance to antibiotics is one of the biggest challenges facing medicine today. Anti-adhesive therapy, using inhibitors of bacterial adhesion to epithelial cells, one of the first stages of infection, is a promising approximation in this area. The size, shape, number of sugar and their placement are variables that have to be taken into account in order to develop multivalent systems able to inhibit the bacterial adhesion based on sugar-lectin interaction. Materials and methods: In the present work we report a modular approach for the synthesis of water-soluble 1D-carbon nanotube-sugar nanoconstructs, with the necessary flexibility to allow an efficient sugar-lectin interaction. The method is based on the reaction of aryl diazonium salts generated in situ from aniline-substituted mannose and lactose derivatives with single wall carbon nanotubes (SWCNTs) sidewalls. Results: Two hybrid nanosystems, I-II, exposing mannose or lactose and having a tetraethylene glycol spacer between the sugar and the nanotube sidewall were rapidly assembled and adequately characterized. The sweet nano-objects were then tested for their ability to agglutinate and selectively inhibit the growth of uropathogenic Escherichia coli. These studies have shown that nanosystem I, exposing mannose on the nanotube surface is able to agglutinate and to inhibit the bacterial growth unlike nano-objects II exposing lactose. Conclusion: The results reported constitute a proof of principle in using mannose-coated 1D-carbon nanotubes as antiadhesive drugs that compete for FimH binding and prevent the uropathogenic bacteria from adhering to the urothelial surface.Andalusian Government (FQM-313 to NK, BIO-026 to REW)Spanish Ministry of Economy and Competitiveness (CTQ2016-78580-C2-1-R to NK)University of Seville (V PLAN PROPIO to REW)European Regional Development Fund (FEDER

An hpr1 point mutation that impairs transcription and mRNP biogenesis without increasing recombination

THO/TREX, a conserved eukaryotic protein complex, is a key player at the interface between transcription and mRNP metabolism. The lack of a functional THO complex impairs transcription, leads to transcriptiondependent hyperrecombination, causes mRNA export defects and fast mRNA decay, and retards replication fork progression in a transcription-dependent manner. To get more insight into the interconnection between mRNP biogenesis and genomic instability, we searched for HPR1 mutations that differentially affect gene expression and recombination. We isolated mutants that were barely affected in gene expression but exhibited a hyperrecombination phenotype. In addition, we isolated a mutant, hpr1-101, with a strong defect in transcription, as observed for lacZ, and a general defect in mRNA export that did not display a relevant hyperrecombination phenotype. In THO single-null mutants, but not in the hpr1 point mutants studied, THO and its subunits were unstable. Interestingly, in contrast to hyperrecombinant null mutants, hpr1-101 did not cause retardation of replication fork progression. Transcription and mRNP biogenesis can therefore be impaired by THO/TREX dysfunction without increasing recombination, suggesting that it is possible to separate the mechanism(s) responsible for mRNA biogenesis defects from the further step of triggering transcriptiondependent recombination.Ministerio de Educación y Ciencia BMC2000-0409 SAF2003-00204Junta de Andalucía CVI10

Sumoylation of Smc5 Promotes Error-free Bypass at Damaged Replication Forks

Replication of a damaged DNA template can threaten the integrity of the genome, requiring the use of various mechanisms to tolerate DNA lesions. The Smc5/6 complex, together with the Nse2/Mms21 SUMO ligase, plays essential roles in genome stability through undefined tasks at damaged replication forks. Various subunits within the Smc5/6 complex are substrates of Nse2, but we currently do not know the role of these modifications. Here we show that sumoylation of Smc5 is targeted to its coiled-coil domain, is upregulated by replication fork damage, and participates in bypass of DNA lesions. smc5-KR mutant cells display defects in formation of sister chromatid junctions and higher translesion synthesis. Also, we provide evidence indicating that Smc5 sumoylation modulates Mph1-dependent fork regression, acting synergistically with other pathways to promote chromosome disjunction. We propose that sumoylation of Smc5 enhances physical remodeling of damaged forks, avoiding the use of a more mutagenic tolerance pathway.Ministerio de Ciencia, Innovacion y Universidades (BFU2015-71308-P, PGC2018-097796-B-I00)AGAUR-Generalitat de Catalunya (2017-SGR-569