Biology subjects

Gasser, S. M.

Publications and source records attributed to Gasser, S. M..

5 recordsLinked to original sources

bioRxiv2021

Destabilization of the holo-DNA Polδ by loss of Pol32 confers conditional lethality that can be suppressed by stabilizing Pol31-Pol3 interaction

DNA Polymerase {delta} plays an essential role in genome replication and in the preservation of genome integrity. In S. cerevisiae, Pol{delta} consists of three subunits: Pol3 (the catalytic subunit), Pol31 and Pol32. We have constructed pol31 mutants by alanine substitution at conserved amino acids, and identified three alleles that do not confer any disadvantage on their own, but which suppress the cold-, HU- and MMS-hypersensitivity of yeast strains lacking Pol32. We have shown that Pol31 and Pol32 are both involved in translesion synthesis, error-free bypass synthesis, and in preservation of replication fork stability under conditions of HU arrest. We identified a solvent exposed loop in Pol31 defined by two alanine substitutions at T415 and W417. Whereas pol31-T4l5A compromises polymerase stability at stalled forks, pol31-W417A is able to suppress many, but not all, of the phenotypes arising from pol32{Delta}. ChIP analyses showed that the absence of Pol32 destabilizes Pole and Pol at stalled replication forks, but does not interfere with checkpoint kinase activation. We show that the Pol31-W417A-mediated suppression of replicationstress sensitivity in pol32{Delta} stems from enhanced interaction between Pol3 and Pol31, which stabilizes a functional Pol{delta}.

molecular biology↗

bioRxiv2020

Uncoordinated long-patch base excision repair at juxtaposed DNA lesions generates a lethal accumulation of double-strand breaks

Inhibition of the TOR pathway (TORC2, or Ypk1/2), or the depolymerization of actin filaments results in catastrophic fragmentation of the yeast genome upon exposure to low doses of the radiomimetic drug Zeocin. We find that the accumulation of double-strand breaks (DSB) is not due to altered DSB repair, but by the uncoordinated activity of base excision repair (BER) at Zeocin-modified DNA bases. We inhibit DSB formation by eliminating glycosylases and/or the endonucleases Apn1/2 and Rad1, implicating these conserved BER enzymes, or events downstream of them, in the conversion of base damage into DSBs. Among DNA polymerases, the reduction of Pol {delta}, and to a lesser extent Pol {varepsilon} and Trf4 (a Pol {beta}-like polymerase), reduces DSB formation. Finally, the BER enzymes, Ogg1 and AP endonuclease, are shown to co-precipitate with actin from yeast extracts and as purified proteins, suggesting that actin may interfere directly with the repair of Zeocin-induced damage.

molecular biology↗

bioRxiv2019

LSM2-8 and XRN-2 contribute to the silencing of H3K27me3-marked genes through targeted RNA decay

In fission yeast and plants, RNA-processing pathways contribute to constitutive and facultative heterochromatin silencing, complementing well-characterized pathways of transcriptional repression. However, it was unclear whether this additional level of regulation occurs in metazoans. Here we describe a pathway of silencing in C. elegans somatic cells, in which the highly conserved, RNA binding complex LSM2-8 selectively silences heterochromatic reporters and endogenous genes bearing the Polycomb mark H3K27me3. Importantly, the LSM2-8 complex works cooperatively with XRN-2, a 5-3 exoribonuclease, and disruption of the pathway leads to mRNA stabilization. This selective LSM2-8-mediated RNA degradation does not target nor depend on H3K9me2/me3, unlike previously described pathways of heterochromatic RNA degradation. Intriguingly, the loss of LSM2-8 coincides with a localized drop in H3K27me3 levels on lsm-8-sensitive loci only. Together this defines a mechanism of RNA degradation that selectively targets a subset of H3K27me3-marked genes, revealing an unrecognized layer of regulation for facultative heterochromatin in animals.

molecular biology↗

bioRxiv2019

Srs2 helicase prevents the formation of toxic DNA damage during late prophase I of yeast meiosis

Proper repair of double-strand breaks (DSBs) is key to ensure proper chromosome segregation. In this study, we found that the deletion of the SRS2 gene, which encodes a DNA helicase necessary for the control of homologous recombination, induces aberrant chromosome segregation during budding yeast meiosis. This abnormal chromosome segregation in srs2 cells accompanies the formation of a novel DNA damage induced during late meiotic prophase-I. The damage may contain long stretches of single-stranded DNAs (ssDNAs), which lead to aggregate formation of a ssDNA binding protein, RPA, and a RecA homolog, Rad51, as well as other recombination proteins inside of the nuclei. The Rad51 aggregate formation in the srs2 mutant depends on the initiation of meiotic recombination and occurs in the absence of chromosome segregation. Importantly, as an early recombination intermediate, we detected a thin bridge of Rad51 between two Rad51 foci or among the foci in the srs2 mutant, which is rarely seen in wild type. These might be cytological manifestation of the connection of two DSB ends and multi-invasion. The DNA damage with Rad51 aggregates in the srs2 mutant is passed through anaphase-I and -II, suggesting the absence of DNA damage-induced cell-cycle arrest after the pachytene stage. We propose that Srs2 helicase resolves early protein-DNA recombination intermediates to suppress the formation of aberrant lethal DNA damage during late prophase-I.

genetics↗

bioRxiv2018

Unexpected erosion of chromosome cohesion by a mitotic prophase-like pathway in yeast meiosis

Sister chromatid cohesion on chromosome arms is essential for the segregation of homologous chromosomes during meiosis I while it is dispensable for sister chromatid separation during mitosis. It was assumed that, unlike the situation in mitosis, chromosome arms retain cohesion prior to onset of anaphase-I. Paradoxically, reduced immunostaining signals of meiosis-specific cohesin, including the kleisin Rec8, from the chromosomes were observed during late prophase-I of budding yeast. This decrease is seen in the absence of Rec8 cleavage and depends on condensin-mediated recruitment of Polo-like kinase (PLK/Cdc5). In this study, we confirmed that this release indeed accompanies the dissociation of acetylated Smc3 as well as Rec8 from meiotic chromosomes during late prophase-I. This release requires, in addition to PLK, the cohesin regulator, Wapl (Rad61/Wpl1 in yeast), and Dbf4-dependent Cdc7 kinase (DDK). Meiosis-specific phosphorylation of Rad61/Wpl1 and Rec8 by PLK and DDK collaboratively promote this release. This process is similar to the vertebrate \"prophase\" pathway for cohesin release during G2 phase and pro-metaphase. In yeast, meiotic cohesin release coincides with PLK-dependent compaction of chromosomes in late meiotic prophase-I. We suggest that yeast uses this highly regulated cleavage-independent pathway to remove cohesin during late prophase-I to facilitate morphogenesis of condensed metaphase-I chromosomes.\n\nAuthor SummaryIn meiosis the life and health of future generations is decided upon. Any failure in chromosome segregation has a detrimental impact. Therefore, it is currently believed that the physical connections between homologous chromosomes are maintained by meiotic cohesin with exceptional stability. Indeed, it was shown that cohesive cohesin does not show an appreciable turnover during long periods in oocyte development. In this context, it was long assumed but not properly investigated, that the prophase pathway for cohesin release would be specific to mitotic cells and will be safely suppressed during meiosis so as not to endanger the valuable chromosome connections. However, a previous study on budding yeast meiosis suggests the presence of cleavage-independent pathway of cohesin release during late prophase-I. In the work presented here we confirmed that the prophase pathway is not suppressed during meiosis, at least in budding yeast and showed that this cleavage-independent release is regulated by meiosis-specific phosphorylation of two cohesin subunits, Rec8 and Rad61(Wapl) by two cell-cycle regulators, PLK and DDK. Our results suggest that late meiotic prophase-I actively controls cohesin dynamics on meiotic chromosomes for chromosome segregation.

cell biology↗