Deletion of the RNR4 gene causes hyperresistance to the carcinogen 4-NQO in the yeast model

La stabilité génomique, qui est essentielle à la vie, est possible grâce à la réplication et la réparation de l’ADN. Une des enzymes responsables de la réplication et de la réparation de l’ADN est la ribonucleotide reductase (RNR), qui est retrouvée chez la levure et chez l’humain. Cette enzyme catalyse la formation de déoxyribonucléotides et maintien le pool de dNTP requis pour la réparation et la réplication de l’ADN. L’enzyme RNR est un tétramère α2β2 constitué d’une grande (R1, α2) et d’une petite (R2, β2) sous-unité. Chez S. cerevisiae, les gènes RNR1 et RNR3 encodent la sous-unité α2 (R1). L’activité catalytique de RNR dépend d’une interaction avec le fer et de la formation d’un complexe entre R1 et R2. L’expression de toutes les sous-unités est inductible par les dommages causés à l’ADN. Dans cette étude, nous démontrons que des cellules qui n’expriment pas une des sous-unités, Rnr4, du complexe RNR sont sensibles à divers agents endommageant l’ADN, tels que le méthyl méthane sulfonate, la bléomycine, le péroxyde d’hydrogène et les rayons ultraviolets (UVC 254 nm). Au contraire, le mutant est résistant au 4-nitroquinoline-1- oxide (4-NQO), un composé qui engendre des lésions encombrantes. Par conséquent, le mutant rnr4Δ démontre une réduction marquée en mutations induites par le 4-NQO comparativement à la souche parentale. Nous voulions identifier la voie de réparation de l’ADN qui conférait cette résistance au 4-NQO ainsi que les protéines impliquées. Les voies BER, NER et MMR n’ont pas aboli la résistance au 4-NQO de la souche rnr4Δ. La protéine recombinante Rad51 ne joue pas un rôle critique dans la réparation de l’ADN et dans la résistance au 4-NQO. La délétion du gène REV3, qui encode une polymérase de contournement, impliquée dans la réparation post-réplication, a partiellement aboli la résistance au 4-NQO dans rnr4Δ. Ces résultats suggèrent que la polymérase Rev3 et possiblement d’autres polymérases translésion (Rev1, Rev7, Rad30) pourraient être impliquées dans la réparation de lésions encombrantes dans l’ADN dans des conditions de carence en dNTP. La réparation de l’ADN, un mécanisme complexe chez la levure, implique une vaste gamme de protéines, dont certaines encore inconnues. Nos résultats indiquent qu’il y aurait plus qu’une protéine impliquée dans la résistance au 4-NQO. Des investigations plus approfondies seront nécessaires afin de comprendre la recombinaison et la réparation post-réplication.Genomic stability, critical for life, is controlled by DNA replication and repair. DNA replication and repair is mediated through many enzymes, one being ribonucleotide reductase (RNR), an enzyme found in both yeast and humans. RNR catalyzes the reaction involved in the formation of deoxyribonucleotides and is responsible for maintaining dNTP pools required for DNA repair and replication. RNR is an α2β2 tetramer consisting of a large (R1, α2) and small subunit (R2, β2). In S. cerevisiae RNR1 and RNR3 encode α2 (R1). RNR catalytic activity depends on its interaction with iron and on the formation of the complex between R1 and R2. All subunits are inducible by DNA damage. Here we show that cells lacking one subunit, Rnr4, of the RNR complex are sensitive to various DNA damaging agents such as methyl methane sulfonate, bleomycin, hydrogen peroxide, and ultraviolet radiation (UVC 254 nm). In contrast, the mutant is resistant to 4-nitroquinoline-1-oxide (4-NQO), an agent which produces bulky lesions. Consistent with this resistance, the rnr4Δ showed a sharp reduction in 4-NQO-induced mutations as compared to the parent. We wanted to determine which pathway was able to confer resistance to 4-NQO and thus targeted DNA repair proteins. The repair pathways BER, NER and MMR did not abolish 4-NQO resistance in rnr4Δ. Recombination protein Rad51 (NHEJ) was lethal in an rnr4Δ thus indicating no role in DNA repair and 4-NQO resistance. Deletion of the REV3 gene, encoding a DNA bypass polymerase involved in post replication repair, partially abolished 4-NQO resistance in rnr4Δ. These results suggest that Rev3 and possibly other translesion polymerases (Rev1, Rev7, Rad30) could play a role in the repair of bulky DNA lesions under low levels of dNTPs. DNA repair, a complex mechanism in yeast, involves a vast array of proteins, some yet to be discovered. Our results indicate that there is more than one protein involved in 4-NQO resistance and further investigation is required concerning recombination and post replication repair

Rrd1 isomerizes RNA polymerase II in response to rapamycin

International audienceBACKGROUND: In Saccharomyces cerevisiae, the immunosuppressant rapamycin engenders a profound modification in the transcriptional profile leading to growth arrest. Mutants devoid of Rrd1, a protein possessing in vitro peptidyl prolyl cis/trans isomerase activity, display striking resistance to the drug, although how Rrd1 activity is linked to the biological responses has not been elucidated.RESULTS: We now provide evidence that Rrd1 is associated with the chromatin and it interacts with RNA polymerase II. Circular dichroism revealed that Rrd1 mediates structural changes onto the C-terminal domain (CTD) of the large subunit of RNA polymerase II (Rpb1) in response to rapamycin, although this appears to be independent of the overall phosphorylation status of the CTD. In vitro experiments, showed that recombinant Rrd1 directly isomerizes purified GST-CTD and that it releases RNA polymerase II from the chromatin. Consistent with this, we demonstrated that Rrd1 is required to alter RNA polymerase II occupancy on rapamycin responsive genes.CONCLUSION: We propose as a mechanism, that upon rapamycin exposure Rrd1 isomerizes Rpb1 to promote its dissociation from the chromatin in order to modulate transcription

Selectivity of antimicrobial peptides: a complex interplay of multiple equilibria

Antimicrobial peptides (AMPs) attack bacterial membranes selectively, killing microbes at concentrations that cause no toxicity to the host cells. This selectivity is not due to interaction with specific receptors, but is determined by the different lipid composition of the membranes of the two cell types, and by the peculiar physico-chemical properties of AMPs, particularly their cationic and amphipathic character. However, the available data, including recent studies of peptide-cell association, indicate that this picture is excessively simplistic, because selectivity is modulated by a complex interplay of several interconnected phenomena. For instance, conformational transitions and self-assembly equilibria modulate the effective peptide hydrophobicity, the electrostatic and hydrophobic contributions to the membrane binding driving force are non-additive, and kinetic processes can play an important role in selective bacterial killing in the presence of host cells. All these phenomena, and their bearing on the final activity and toxicity of AMPs, must be considered in the definition of design principles to optimize peptide selectivity