Biology subjects

Rosas Bringas, F. R.

Publications and source records attributed to Rosas Bringas, F. R..

7 recordsLinked to original sources

bioRxiv2025

The shortest telomere in telomerase-negative cells triggers replicative senescence at a critical threshold length and also fuels genomic instability

In the absence of telomerase, telomere shortening triggers replicative senescence, a tumor suppressor mechanism that is also associated with oncogenic genomic instability. Yet, the precise mechanism that connects these seemingly opposing forces remains poorly understood. To directly study the complex interplay between senescence, telomere dynamics, and genomic instability, we developed a system in Saccharomyces cerevisiae to generate and track telomeres of precise length in the absence of telomerase. Using single-telomere and single-cell analyses combined with mathematical modeling, we identify a threshold length at which telomeres switch into dysfunction. A single shortest telomere below the threshold length is necessary and sufficient to trigger the onset of replicative senescence in a majority of cells. At population level, fluctuation assays establish that rare genomic instability arises predominantly in cis to the shortest telomere as Pol32-dependent non-reciprocal translocations that result in re-elongation of the shortest telomere and likely transient escape from senescence. The switch of the shortest telomere into dysfunction and subsequent processing in telomerase-negative cells thus serves as the mechanistic link between replicative senescence onset, genomic instability and the initiation of post-senescence survival.

molecular biology↗

bioRxiv2024

Interstitial telomeric sequences promote gross chromosomal rearrangement via multiple mechanisms

Telomeric DNA sequences are difficult to replicate. Replication forks frequently pause or stall at telomeres, which can lead to telomere truncation and dysfunction. In addition to being at chromosome ends, telomere repeats are also present at internal locations within chromosomes, known as interstitial telomeric sequences (ITSs). These sequences are unstable and prone to triggering gross chromosomal rearrangements (GCRs). In this study, we quantitatively examined the effect of ITSs on GCR rate in Saccharomyces cerevisiae using a genetic assay. We find that GCR rate increases exponentially with ITS length. This increase can be attributed to the telomere repeat binding protein Rap1 impeding DNA replication and a bias of repairing DNA breaks at or distal to the ITS via de novo telomere addition. Additionally, we performed a genome-wide screen for genes that modulate the rate of ITS-induced GCRs. We find that mutation of core components of the DNA replication machinery leads to an increase in GCRs, but many mutants known to increase GCR rate in the absence of an ITS do not significantly affect GCR rate when an ITS is present. We also identified genes that promote the formation of ITS-induced GCRs, including genes with roles in telomere maintenance, nucleotide excision repair, and transcription. Our work thus uncovers multiple mechanisms by which an ITS promotes GCR. Significance statementTelomeric DNA repeats are found at the ends of linear chromosomes where they, together with specialized proteins that bind to them, protect chromosome ends from degradation and unwanted DNA repair activities. Telomeric repeats can also be found at internal locations in the genome, where they are called interstitial telomeric sequences (ITSs). ITSs are prone to breakage and are associated with human diseases. In this study, using bakers yeast as a model organism, we show that instability at ITSs is driven by multiple factors, and identify genes that either promote or suppress gross chromosomal rearrangements induced by the presence of an ITS.

molecular biology↗

bioRxiv2024

Revisiting the role of the spindle assembly checkpoint in the formation of gross chromosomal rearrangements in Saccharomyces cerevisiae

Multiple pathways are known to suppress the formation of gross chromosomal rearrangements (GCRs), which can cause human diseases including cancer. In contrast, much less is known about pathways that promote their formation. The spindle assembly checkpoint (SAC), which ensures the proper separation of chromosomes during mitosis, has been reported to promote GCR, possibly by delaying mitosis to allow GCR-inducing DNA repair to occur. Here we show that this conclusion is the result of an experimental artifact arising from the synthetic lethality caused by disruption of the SAC and loss of the CIN8 gene, which is often lost in the genetic assay used to select for GCRs. After correcting for this artifact, we find no role of the SAC in promoting GCR. Significance statementA gross chromosomal rearrangement (GCR) is an abnormal structural change of a native chromosome. Examples of GCRs include deletions, duplications, inversions, and translocations. GCRs can lead to genetic diseases such as cancer. A previous study implicated the spindle assembly checkpoint (SAC), which ensures the proper separation of chromosomes during cell division, in facilitating the formation of GCRs. In this study, we show that this is not the case; the SAC does not promote GCR.

molecular biology↗

bioRxiv2023

Deletion of MEC1 suppresses replicative senescence of the cdc13-2 mutant in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, telomerase recruitment to telomeres depends on a direct interaction between Cdc13, a protein that binds single-stranded telomeric DNA, and the Est1 subunit of telomerase. The cdc13-2 allele disrupts telomerase association with telomeres, resulting in progressive telomere shortening and replicative senescence. The Mec1/ATR kinase is both a positive and negative regulator of telomerase activity, and is required for the cell cycle arrest in telomerase-deficient senescent cells. In this study, we find that deletion of MEC1 suppresses the replicative senescence of cdc13-2. This suppression is dependent on telomerase, indicating that Mec1 antagonizes telomerase-mediated telomere extension in cdc13-2 cells to promote senescence.

molecular biology↗

bioRxiv2021

A synthetic lethal screen identifies HDAC4 as a potential target in MELK overexpressing cancers

Maternal embryonic leucine zipper kinase (MELK) is frequently overexpressed in cancer, but the role of MELK in cancer is still poorly understood. MELK was shown to have roles in many cancer-associated processes including tumor growth, chemotherapy resistance, and tumor recurrence. To determine whether the frequent overexpression of MELK can be exploited in therapy, we performed a high-throughput screen using a library of Saccharomyces cerevisiae mutants to identify genes whose functions become essential when MELK is overexpressed. We identified two such genes: LAG2 and HDA3. LAG2 encodes an inhibitor of the SCF ubiquitin-ligase complex, while HDA3 encodes a subunit of the HDA1 histone deacetylase complex. We find that one of these synthetic lethal interactions is conserved in mammalian cells, as inhibition of a human homolog of HDA3 (HDAC4) is synthetically toxic in MELK overexpression cells. Altogether, our work might provide a new angle of how to exploit MELK overexpression in cancers and might thus lead to novel intervention strategies.

genetics↗

bioRxiv2021

Suppression of cdc13-2-associated senescence by pif1-m2 requires Ku-mediated telomerase recruitment

In Saccharomyces cerevisiae, recruitment of telomerase to telomeres requires an interaction between Cdc13, which binds single-stranded telomeric DNA, and the Est1 subunit of telomerase. A second pathway involving an interaction between the yKu complex and telomerase RNA (TLC1) contributes to telomerase recruitment, but cannot sufficiently recruit telomerase on its own to prevent replicative senescence when the primary Cdc13-Est1 pathway is abolished--for example, in the cdc13-2 mutant. In this study, we find that mutation of PIF1, which encodes a helicase that inhibits telomerase, suppresses the replicative senescence of cdc13-2 increasing reliance on the yKu-TLC1 pathway for telomerase recruitment. Our findings reveal new insight into telomerase-mediated telomere maintenance.

molecular biology↗

bioRxiv2020

Rap1, Rif2, and the Ku complex work in concert to cap chromosome ends

Rap1 is the main protein that binds double-stranded telomeric DNA in Saccharomyces cerevisiae. Examination of the telomere functions of Rap1 is complicated by the fact that it also acts as a transcriptional regulator of hundreds of genes and is encoded by an essential gene. In this study, we disrupt Rap1 telomere association by expressing a mutant telomerase RNA subunit (tlc1-tm) that introduces mutant telomeric repeats. tlc1-tm cells grow similar to wild-type cells, although depletion of Rap1 at telomeres causes defects in telomere length regulation and telomere capping. Rif2 is a protein normally recruited to telomeres by Rap1, but we show that Rif2 can still associate with Rap1-depleted tlc1-tm telomeres, and that this association is required to inhibit telomere degradation by the MRX complex. We find that Rap1, Rif2, and the Ku complex work in parallel to prevent telomere degradation, and absence of all three at telomeres causes lethality. The partially redundant mechanisms may explain the rapid evolution of telomere components in budding yeast species.

molecular biology↗