Biology subjects

Waterman, D. P.

Publications and source records attributed to Waterman, D. P..

4 recordsLinked to original sources

bioRxiv2025

Esc1-mediated anchoring regulates telomere clustering in response to metabolic changes.

Spatial organization of yeast telomeres is highly dynamic and regulated by growth conditions. In rich medium, the 32 telomeres group in 3 to 5 foci at the nuclear periphery. This organization is drastically rearranged in long-lived quiescent (Q) cells forming upon carbon source exhaustion: telomere foci assemble into a hypercluster containing most telomeres and located in the center of the nucleus contributing to their long-term viability. Here we explore the mechanisms regulating telomere distribution during this transition. We rule out a modification of telomere-telomere interactions via the telomeric protein Sir3 as the main factor regulating hypercluster formation. However, our physical modeling predicts that telomere anchoring antagonizes telomere clustering. Systematic deletion of all known telomere anchors identifies the inner nuclear membrane-associated protein Esc1, as a key telomeric anchor after the diauxic shift. Our data support a model where Esc1 mediated anchoring is progressively lost upon entry into quiescence through dephosphorylation of a single residue of Esc1, resulting in the formation of telomere hyperclusters in the center of the nucleus in Q cells.

cell biology↗

bioRxiv2023

Prolonged DNA Damage Checkpoint Arrest Requires Constant Renewal and the Spindle Assembly Checkpoint

Cells evoke the DNA damage checkpoint (DDC) to inhibit mitosis in the presence of DNA double-strand breaks (DSBs) to allow more time for DNA repair. In budding yeast, a single irreparable DSB is sufficient to activate the DDC and induce cell cycle arrest prior to anaphase for about 12 to 15 hours, after which cells "adapt" to the damage by extinguishing the DDC and resuming the cell cycle. While activation of the DNA damage-dependent cell cycle arrest is well-understood, how it is maintained remains unclear. To address this, we conditionally depleted key DDC proteins after the DDC was fully activated and monitored changes in the maintenance of cell cycle arrest. Degradation of Ddc2ATRIP, Rad9, Rad24, or Rad53CHK2 results in premature resumption of the cell cycle, indicating that these DDC factors are required both to establish and to maintain the arrest. Dun1 is required for establishment, but not maintenance of arrest, whereas Chk1 is required for prolonged maintenance but not for initial establishment of the mitotic arrest. When the cells are challenged with 2 persistent DSBs, they remain permanently arrested. This permanent arrest is initially dependent on the continuous presence of Ddc2, Rad9, and Rad53; however, after 15 hours these proteins become dispensable. Instead, the continued mitotic arrest is sustained by spindle-assembly checkpoint (SAC) proteins Mad1, Mad2, and Bub2 but not by Bub2s binding partner Bfa1. These data suggest that prolonged cell cycle arrest in response to 2 DSBs is achieved by a handoff from the DDC to specific components of the SAC. Furthermore, the establishment and maintenance of DNA damage-induced cell cycle arrest requires overlapping but different sets of factors.

genetics↗

bioRxiv2019

Yeast Securin is trafficked through the endomembrane system to regulate cell cycle arrest during the DNA damage response

The yeast securin protein, Pds1, belongs to a class of highly conserved eukaryotic proteins that regulate the timing of chromatid segregation during mitosis by inhibiting separase, Esp1. During the metaphase to anaphase transition Pds1 is degraded by the ubiquitin-proteasome system in the nucleus, unleashing Esp1s protease activity to cleave cohesin surrounding sister chromatids. In response to DNA damage Pds1 is phosphorylated and stabilized, stalling mitotic progression to preserve genomic integrity. In addition, during the DNA damage checkpoint response, securin and separase are partially localized in the vacuole. Here we genetically dissect the requirements for securins vacuolar localization and find that it is dependent on the Alkaline Phosphatase (ALP) endosomal transport pathway but not autophagy. Blocking retrograde traffic between the Golgi and the endoplasmic reticulum, by inhibiting COPI vesicle transport, drives Pds1 into the vacuole, whereas inhibiting antegrade transport by disrupting COPII-mediated traffic, results in the unexpected loss of Pds1 and the extinction of DNA damage-induced cell cycle arrest. We report that the induction of ER stress, either genetically or by treating cells with dithiothreitol, is sufficient to extinguish the DNA damage checkpoint, suggesting crosstalk between these two pathways. These data highlight new ways in which Pds1 and Esp1 are regulated during a DNA damage induced G2/M arrest and its requirement in the maintenance of the DNA damage checkpoint response.

cell biology↗

bioRxiv2017

CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles

Harnessing CRISPR-Cas9 technology has provided an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We have analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in the budding yeast Saccharomyces cerevisiae and find that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a one base pair (+1) insertion that appears to result from Cas9 creating a 1-bp 5 overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which were biased toward homonucleotide insertions. Using inverted PAM (iPAM) sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9::gRNA complex influences subsequent NHEJ processing. As with well-studied events induced by the site-specific HO endonuclease, CRISPR-Cas9 mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events, however, are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11s nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells (van Overbeek et al., 2016) reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

molecular biology↗