Translesion synthesis DNA polymerase η exhibits a specific RNA extension activity and a transcription-associated function

We thank Andres Aguilera for providing the pCYC-LacZ plasmid for the GLRO experiments, and Szilvia Minorits for technical assistance. This work was also supported by grants from the National Research, Development and Innovation Office: GINOP-2.3.2-15-2016-00001 and GINOP-2.3.2-15-2016-00024.Peer reviewedPublisher PD

The Zn-Finger of Saccharomyces cerevisiae Rad18 and Its Adjacent Region Mediate Interaction with Rad5

DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structure-function based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways

Structural conservation of insulin/IGF signalling axis at the insulin receptors level in Drosophila and humans

The insulin-related hormones regulate key life processes in Metazoa, from metabolism to growth, lifespan and aging, through an evolutionarily conserved insulin signalling axis (IIS). In humans the IIS axis is controlled by insulin, two insulin-like growth factors, two isoforms of the insulin receptor (hIR-A and -B), and its homologous IGF-1R. In Drosophila, this signalling engages seven insulin-like hormones (DILP1-7) and a single receptor (dmIR). This report describes the cryoEM structure of the dmIR ectodomain:DILP5 complex, revealing high structural homology between dmIR and hIR. The excess of DILP5 yields dmIR complex in an asymmetric 'T' conformation, similar to that observed in some complexes of human IRs. However, dmIR binds three DILP5 molecules in a distinct arrangement, showing also dmIR-specific features. This work adds structural support to evolutionary conservation of the IIS axis at the IR level, and also underpins a better understanding of an important model organism

Mutations at the Subunit Interface of Yeast Proliferating Cell Nuclear Antigen Reveal a Versatile Regulatory Domain

Acknowledgments We thank Szilvia Minorits for technical assistance. I.U. conceived and designed the project and wrote the manuscript. All authors participated in designing and performing the experiments, and analyzing the results. The authors declare no competing financial interests. This work was also supported by a grant from the National Research, Development and Innovation Office GINOP-2.3.2-15-2016-00001. Funding: This work was supported by Hungarian Science Foundation Grant OTKA 109521 and National Research Development and Innovation Office GINOP-2.3.2-15-2016-00001. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Spontaneous mutation rates in yeast strains expressing the PCNA variants.

<p>Spontaneous mutation rates in yeast strains expressing the PCNA variants.</p

Percentage of cells displaying the large budded (dumbbell) morphology of yeast strains expressing the PCNA variants.

<p>Percentage of cells displaying the large budded (dumbbell) morphology of yeast strains expressing the PCNA variants.</p

Phenotypic characterization of the mutants.

<p>(A) Growth of the strains at different temperatures. Approximately 100 cells of each strain were plated on rich medium and the plates were incubated at the indicated temperatures for 2 days. (B) Sensitivity of the strains to HU. Ten fold serial dilutions of the indicated strains were spotted on YPD plates containing the given amount of HU. (C) Sensitivity of the strains to DNA damaging agents. Cells were spotted as described for (B) on plates either exposed to the indicated UV dose, or containing the indicated amount of MMS. For (B) and (C) several HU, UV, and MMS doses were applied, but only plates with the most appropriate doses are presented. (D) Quantitative assays of UV and MMS induced killing of the indicated strains. The results represent the average of three experiments. Standard deviations are indicated.</p

In the II99,100AA PCNA mutant strain the <i>REV3</i> branch of translesion synthesis is inactivated.

<p>(A-B) The II99,100AA PCNA mutant shows epistasis with <i>RAD18</i> upon UV treatment. (C-D) The II99,100AA PCNA mutant affects the Rev3 pathway. On (A) and (C) ten fold serial dilutions of cells with the indicated genotypes were spotted on plates and exposed to UV doses indicated on the right. (B) and (D) show quantitative analysis of UV-survival of the indicated strains. (E) UV-induced mutagenesis is abolished in the II99,100AA PCNA mutant strain. Forward mutation rates at the <i>CAN1</i> locus were determined after exposing the cells to the indicated UV doses. (B), (D), and (E) represent the average of three experiments. Standard deviations are indicated. P-values representing the significance of difference are also shown on (E). *:p<0.05 ** p<0.01, *** p<0.001 ns: no statistical difference.</p