Biology subjects

Kupiec, M.

Publications and source records attributed to Kupiec, M..

6 recordsLinked to original sources

bioRxiv2025

Calcium signaling is a universal carbon source signal transducer and effects an ionic memory of past carbon sources

Glucose is the preferred carbon source for most cells. However, cells may encounter other carbon sources that can be utilized. How cells match their metabolic gene expression to their carbon source, beyond a general glucose repressive system (catabolite repression), remains little understood. By studying the effect of up to seven different carbon sources on Snf1 phosphorylation and on the expression of downstream regulated genes, we searched for the mechanism that identifies carbon sources. We found that the glycolysis metabolites glucose-6-phosphate (G6P) and glucose-1-phosphate (G1P) play a central role in the adaptation of gene expression to different carbon sources. The ratio of G1P and G6P activates an analogue calcium signaling via the proton-exporter Pma1, to regulate downstream genes. The signaling pathway bifurcates with calcineurin reducing ADH2 (alcohol dehydrogenase) expression and with Cmk1 increasing ZWF1 (glucose-6-phosphate dehydrogenase) expression. Furthermore, calcium signaling is not only regulated by the present carbon source; it is also regulated by past carbon sources. We were able to manipulate this ionic memory mechanism to obtain high expression of ZWF1 in media containing galactose. Our findings provide a universal mechanism by which cells respond to all carbon sources.

molecular biology↗

bioRxiv2024

PCNA unloading is crucial for the bypass of DNA lesions by homologous recombination

The DNA Damage Tolerance (DDT) mechanisms allow cells to bypass lesions in the DNA during replication. This allows the cells progress normally through the cell cycle in face of abnormalities in their DNA. PCNA, a homotrimeric sliding clamp complex plays a central role in the coordination of various processes during DNA replication, including the choice of mechanism used during DNA damage bypass. Mono-or poly-ubiquitination of PCNA facilitate an error-prone or an error-free bypass mechanism, respectively. In contrast, SUMOylation recruits the Srs2 helicase, which prevents local homologous recombination. The Elg1 RFC-like complex plays an important role in unloading PCNA from the chromatin. We analyse the interaction of mutations that destabilize PCNA with mutations in the Elg1 clamp unloader and the Srs2 helicase. Our results suggest that in addition to its role as a coordinator of bypass mechanisms, the very presence of PCNA on the chromatin prevents homologous recombination, even in the absence of the Srs2 helicase. Thus, PCNA unloading seems to be a pre-requisite for recombinational repair.

genetics↗

bioRxiv2023

Control of telomere length in yeast by SUMOylated PCNA and the Elg1 PCNA unloader

Telomeres cap and protect the linear eukaryotic chromosomes. Telomere length is determined by an equilibrium between positive and negative regulators of telomerase activity. A systematic screen for yeast mutants that affect telomere length maintenance in the yeast Saccharomyces cerevisiae revealed that mutations in any of [~]500 genes affects telomere length. One of the genes that, when mutated, causes telomere elongation is ELG1, which encodes an unloader of PCNA, the processivity factor for replicative DNA polymerases. PCNA can undergo SUMOylation on two conserved residues, K164 and K127, or ubiquitination at lysine 164. These modifications have already been implicated in genome stability processes. We report that SUMOylated PCNA acts as a signal that positively regulates telomerase activity. We also uncovered physical interactions between Elg1 and the CST (Cdc13-Stn1-Ten) complex, and dissected the mechanism by which Elg1 and Stn1 negatively regulates telomere elongation, coordinated by SUMO. We present a model that provides mechanistic insights on how chromosomal replication and telomere elongation are coordinated.

molecular biology↗

bioRxiv2022

S. cerevisiae cells can grow without the Pds5 cohesin subunit

During DNA replication, the newly created sister chromatids are held together until their separation at anaphase. The cohesin complex is in charge of creating and maintaining sister-chromatid cohesion (SCC) in all eukaryotes. In S. cerevisiae cells, cohesin is composed of two elongated proteins, Smc1 and Smc3, bridged by the kleisin Mcd1/Scc1. The latter also acts as a scaffold for three additional proteins, Scc3/Irr1, Wpl1/Rad61, and Pds5. Although the HEAT-repeat protein Pds5 is essential for cohesion, its precise function is still debated. Deletion of the ELG1 gene, encoding a PCNA unloader, can partially suppress the temperature-sensitive pds5-1 allele, but not a complete deletion of PDS5. We carried out a genetic screen for high copy number suppressors and another for spontaneously arising mutants, allowing the survival of a pds5{Delta} elg1{Delta} strain. Our results show that cells remain viable in the absence of Pds5 provided that there is both an elevation in the level of Mcd1 (which can be due to mutations in the CLN2 gene, encoding a G1 cyclin), and an increase in the level of SUMO-modified PCNA on chromatin (caused by lack of PCNA unloading in elg1{Delta} mutants). The elevated SUMO-PCNA levels increase the recruitment of the Srs2 helicase, which evicts Rad51 molecules from the moving fork, creating ssDNA regions that serve as sites for increased cohesin loading and SCC establishment. Thus, our results delineate a double role for Pds5 in protecting the cohesin ring and interacting with the DNA replication machinery. IMPORTANCESister chromatid cohesion is vital for faithful chromosome segregation, chromosome folding into loops, and gene expression. A multisubunit protein complex known as cohesin holds the sister chromatids from S-phase until the anaphase stage. In this study, we explore the function of the essential cohesin subunit Pds5 in the regulation of sister chromatid cohesion. We performed two independent genetic screens to bypass the function of the Pds5 protein. We observe that Pds5 protein is a cohesin stabilizer, and elevating the levels of Mcd1 protein along with SUMO-PCNA accumulation on chromatin can compensate for the loss of the PDS5 gene. In addition, Pds5 plays a role in coordinating the DNA replication and sister chromatid cohesion establishment. This work elucidates the function of cohesin subunit Pds5, the G1 cyclin Cln2, and replication factors PCNA, Elg1 and Srs2 in the proper regulation of sister chromatid cohesion.

genetics↗

bioRxiv2020

Access to PCNA by Srs2 and Elg1 controls the choice between alternative repair pathways in yeast

During DNA replication stalling can occur when the replicative DNA polymerases encounter lesions or hard-to replicate regions. Under these circumstances the processivity factor PCNA gets ubiquitylated at lysine 164, inducing the DNA damage tolerance (DDT) mechanisms that can bypass lesions encountered during DNA replication. PCNA can also be SUMOylated at the same residue or at lysine 127. Surprisingly, pol30-K164R mutants display a higher degree of sensitivity to DNA damaging agents than pol30-KK127,164RR strains, unable to modify any of the lysines. Here we show that in addition to trans-lesion synthesis and strand-transfer DTT mechanisms, an alternative repair mechanism ("salvage recombination") that copies information from the sister chromatid, is repressed by the recruitment of Srs2 to SUMOylated PCNA. Overexpression of Elg1, the PCNA unloader, or of the recombination protein Rad52 allows its activation. We dissect the genetic requirements for this pathway, as well as the interactions between Srs2 and Elg1.

genetics↗

bioRxiv2017

Pivotal roles of PCNA loading and unloading on heterochromatin function

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 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.\n\nSIGNIFICANCE STATEMENTDNA replication poses a unique logistical challenge for the cell in that structural features of chromatin and their regulatory functions must be carefully coordinated with passage of replication machinery so faithful duplication of both the genome and its chromatin structures may be achieved. Nucleosome assembly is fundamental to reestablishment of chromatin in the wake of DNA replication, and here a mechanism by which nucleosome assembly is coordinated with DNA replication to maintain silenced chromatin is described.

genetics↗