Biology subjects

Lisby, M.

Publications and source records attributed to Lisby, M..

11 recordsLinked to original sources

bioRxiv2022

CRI-SPA, a mating based CRISPR-Cas9 assisted method for high-throughput genetic modification of yeast strain libraries

AbstractBiological functions are orchestrated by intricate networks of interacting genetic elements. Predicting the interaction landscape remains a challenge for systems biology and the identification of phenotypic maximas would be of great benefit to synthetic biology. Thus, new research tools allowing simple and rapid mapping of sequence to function are required to forward these research fields. Here, we describe CRI-SPA, a method allowing the transfer of a chromosomal genetic feature from a donor strain to arrayed strains in large libraries of Saccharomyces cerevisiae. CRI-SPA is based on mating, CRISPR-Cas9-induced gene conversion and Selective Ploidy Ablation and is executed within a week. We demonstrate the power of CRI-SPA by transferring four genes responsible for the production of betaxanthin, a yellow biosensor for the morphine precursor L-DOPA, into each strain of the yeast knock-out collection ({approx}4800 strains), providing a genome-wide overview of the genetic requirements for betaxanthin production. CRI-SPA is fast, highly reproducible, can be massively parallelized with automation and does not require selection for the transferred genetic feature.

systems biology↗

bioRxiv2022

Rdh54 stabilizes Rad51 at displacement loop intermediates to regulate genetic exchange between chromosomes

Homologous recombination (HR) is a double-strand break DNA repair pathway that preserves chromosome structure. To repair the damaged recipient DNA, HR requires an intact donor DNA sequence located elsewhere in the genome. After the double-strand break is repaired, DNA sequence information can be transferred between donor and recipient DNA molecules through different mechanisms, including DNA crossovers that form between homologous chromosomes. Regulating this transfer of information is an important step in effectively completing HR and maintaining genome integrity. For example, mitotic exchange of information between homologous chromosomes can result in loss-of-heterozygosity (LOH) in diploid organisms, and in higher eukaryotes, the development of cancer. The DNA motor protein Rdh54 is a highly conserved DNA translocase that functions during HR but has a limited role in repairing DNA. Instead, several existing phenotypes in rdh54{Delta} strains suggest that Rdh54 may regulate the flow of information between donor and recipient DNA molecules post DNA repair. In our current study, we used a combination of biochemical and genetic techniques to dissect the role of Rdh54 on the exchange of genetic information after DNA repair. Our data indicates that RDH54 regulates DNA sequence exchange between chromosomes by limiting the disruption of Rad51 at an early HR intermediate called the displacement loop (D-loop). Rdh54 also protects Rad51 filaments, acting in opposition to Rad51 removal by the DNA motor protein Rad54. Furthermore, we find that expression of a catalytically inactivate allele of Rdh54, rdh54K318R, displays a different distribution of information exchange outcomes than rdh54{Delta} cells. From these results, we propose a model for how Rdh54 may effectively regulate information transfer during homologous recombination.

biochemistry↗

bioRxiv2022

A conserved PLK1 docking site in TopBP1 maintains genome integrity during mitosis

TopBP1 is a large scaffold protein with multiple functions in genome integrity. We previously identified a novel role for TopBP1 during M phase by showing that TopBP1 reduces carry-over of DNA damage to daughter cells. This function emerges as a critical backup pathway in BRCA deficient cells, yet many aspects of TopBP1 regulation during mitosis are unclear. The mitotic kinase PLK1 has been reported to interact with TopBP1 but the functional relevance of this is unclear. Here, we identify and characterize a conserved PLK1 docking site in TopBP1. Endogenous deletion of the PLK1 docking site in TopBP1 results in increased number of mitotic TopBP1 foci, increased DNA damage in daughter cells, deficient mitotic DNA repair synthesis and increased frequency of binucleation. At the same time, cell cycle distribution and ATR activation are normal in cells with the PLK1 docking site deletion in TopBP1. Interestingly, mutation of this site in TopBP1 renders cells sensitive to PARP inhibitors but not to camptothecin hinting to different cellular effects of the two chemotherapeutics. Altogether, our data indicate that the PLK1-TopBP1 interaction is critical for the mitotic function of TopBP1.

cell biology↗

bioRxiv2021

Cdc48 targets INQ-localized Mrc1 to facilitate recovery from replication stress

DNA replication stress is a source of genome instability and a replication checkpoint has evolved to enable fork stabilisation and completion of replication during stress. Mediator of the replication checkpoint 1 (Mrc1) is the primary mediator of this response in Saccharomyces cerevisiae. Mrc1 is partially sequestered in the intranuclear quality control compartment (INQ) upon methyl methanesulfonate (MMS)-induced replication stress. Here we show that Mrc1 re-localizes from the replication fork to INQ during replication stress. Sequestration of Mrc1 in INQ is facilitated by the Btn2 chaperone and the Cdc48 segregase is required to release Mrc1 from INQ during recovery from replication stress. Consistently, we show that Cdc48 colocalizes with Mrc1 in INQ and we find that Mrc1 is recognized by the Cdc48 cofactors Ufd1 and Otu1, which contribute to clearance of Mrc1 from INQ. Our findings suggest that INQ localization of Mrc1 and Cdc48 function to facilitate replication stress recovery by transiently sequestering the replication checkpoint mediator Mrc1 and explains our observation that Btn2 and Cdc48 are required for efficient replication restart following MMS-induced replication stress.

cell biology↗

bioRxiv2021

A complex of BRCA2 and PP2A-B56 is required for DNA repair by homologous recombination

Mutations in the tumour suppressor gene BRCA2 are associated with predisposition to breast and ovarian cancers. BRCA2 has a central role in maintaining genome integrity by facilitating the repair of toxic DNA double-strand breaks (DSBs) by homologous recombination (HR). BRCA2 acts by promoting RAD51 nucleoprotein filament formation on resected single-stranded DNA, but how BRCA2 activity is regulated during HR is not fully understood. Here, we delineate a pathway where ATM and ATR kinases phosphorylate a highly conserved region in BRCA2 in response to DSBs. These phosphorylations stimulate the binding of the protein phosphatase PP2A-B56 to BRCA2 through a conserved binding motif. We show that the phosphorylation-dependent formation of the BRCA2-PP2A-B56 complex is required for efficient RAD51 loading to sites of DNA damage and HR-mediated DNA repair. Moreover, we find that several cancer-associated mutations in BRCA2 deregulate the BRCA2-PP2A-B56 interaction and sensitize cells to PARP inhibition. Collectively, our work uncovers PP2A-B56 as a positive regulator of BRCA2 function in HR with clinical implications for BRCA2 and PP2A-B56 mutated cancers.

cell biology↗

bioRxiv2021

Large field of view-spatially resolved transcriptomics at nanoscale resolution

Spatially resolved transcriptomic technologies are promising tools to study cell fate decisions in a physical microenvironment, which is fundamental for enhancing our knowledge of mammalian development. However, the imbalance between resolution, transcript capture and field of view of current methodologies precludes their systematic application to analyze relatively large and three-dimensional mid- and late-gestation mammalian embryos. Here, we combined DNA nanoball (DNB) patterned arrays and tissue RNA capture to create SpaTial Enhanced REsolution Omics-sequencing (Stereo-seq). This approach allows transcriptomic profiling of large histological sections with high resolution and sensitivity. We have applied Stereo-seq to study the kinetics and directionality of transcriptional variation in a time course of mouse organogenesis. We used this information to gain insight into the molecular basis of regional specification, neuronal migration and differentiation in the developing brain. Furthermore, we mapped the expression of a panel of developmental disease-related loci on our global transcriptomic maps to define the spatiotemporal windows of tissue vulnerability. Our panoramic atlas constitutes an essential resource to investigate longstanding questions concerning normal and abnormal mammalian development.

cell biology↗

bioRxiv2020

Rad51 paralog complex Rad55-Rad57 acts as a molecular chaperone during homologous recombination

Homologous recombination (HR) is essential for the maintenance of genome integrity. Rad51 paralogs fulfill a conserved, but undefined role in HR, and their mutations are associated with increased cancer risk in humans. Here, we use single-molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55-Rad57 promotes the assembly of Rad51 recombinase filaments through transient interactions, providing evidence that it acts as a classical molecular chaperone. Srs2 is an ATP-dependent anti-recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55- Rad57 does not physically block the movement of Srs2. Instead, Rad55-Rad57 promotes rapid re- assembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and ssDNA-bound states, the rate of which is dynamically controlled though the opposing actions of Rad55-Rad57 and Srs2.

biophysics↗

bioRxiv2020

Topoisomerase II deficiency leads to a postreplicative structural shift in all Saccharomyces cerevisiae chromosomes.

The key role of Topoisomerase II (Top2) is the removal of topological intertwines between sister chromatids. In yeast, inactivation of Top2 brings about distinct cell cycle responses. In the case of the conditional top2-5 allele, interphase and mitosis progress on schedule but cells suffer from a segregation catastrophe. We here show that top2-5 chromosomes fail to enter a Pulsed-Field Gel Electrophoresis (PFGE) in the first cell cycle, a behavior traditionally linked to the presence of replication and recombination intermediates. We distinguished two classes of affected chromosomes: the rDNA-bearing chromosome XII, which fails to enter a PFGE at the beginning of S-phase, and all the other chromosomes, which fail at a postreplicative stage. In synchronously cycling cells, this late PFGE retention is observed in anaphase; however, we demonstrate that this behavior is independent of cytokinesis, stabilization of anaphase bridges, spindle pulling forces and even anaphase onset. Strikingly, once the PFGE retention has occurred it becomes refractory to Top2 re-activation. DNA combing, two-dimensional electrophoresis, genetic analyses and GFP-tagged DNA damage markers suggest that non-recombinational modifications of late replication intermediates may account for the shift in the PFGE behavior. The fact that this shift does not trigger G2/M checkpoints further supports this statement since checkpoints are active for other replicative stresses in the absence of Top2. We propose that the prolonged absence of Top2 activity leads to a general chromosome structural change. This change might interlock chromatids together with catenations and thus contribute to the formation of anaphase bridges in top2 mutants.

molecular biology↗

bioRxiv2020

Folliculin variants linked to Birt-Hogg-Dube syndrome are targeted for proteasomal degradation

Germline mutations in the folliculin (FLCN) tumor suppressor gene are linked to Birt-Hogg-Dube (BHD) syndrome, a dominantly inherited genetic disease characterized by predisposition to fibrofolliculomas, lung cysts, and renal cancer. Most BHD-linked FLCN variants include large deletions and splice site aberrations predicted to cause loss of function. The mechanisms by which missense variants and short in-frame deletions in FLCN trigger disease are unknown. Here, we present computational and experimental studies showing that the majority of such disease-causing FLCN variants cause loss of function due to proteasomal degradation of the encoded FLCN protein, rather than directly ablating FLCN function. Accordingly, several different single-site FLCN variants are present at strongly reduced levels in cells. In line with our finding that FLCN variants are protein quality control targets, several are also highly insoluble and fail to associate with the FLCN-binding partners FNIP1 and FNIP2. The lack of FLCN binding leads to rapid proteasomal degradation of FNIP1 and FNIP2. Half of the tested FLCN variants are mislocalized in cells, and one variant ({Delta}E510) forms perinuclear protein aggregates. A yeast-based screen revealed that the deubiquitylating enzyme Ubp15/USP7 and molecular chaperones regulate the turnover of the FLCN variants. Lowering the temperature to 29 {degrees}C led to a stabilization of two FLCN missense proteins, and for one variant (R362C), FLCN function was re-established at low temperature. In conclusion, we propose that most BHD-linked FLCN missense variants and small in-frame deletions operate by causing misfolding and degradation of the FLCN protein, and that stabilization of certain disease-linked variants may hold therapeutic potential.

molecular biology↗

bioRxiv2018

A critical role for Dna2 at unwound telomeres

Dna2 is a nuclease and helicase that functions redundantly with other proteins in Okazaki fragment processing, double strand break (DSB) resection and checkpoint kinase activation. Dna2 is an essential enzyme, required for yeast and mammalian cell viability. Here we report that numerous mutations affecting the DNA damage checkpoint suppress dna2{Delta} lethality in Saccharomyces cerevisiae. dna2{Delta} cells are also suppressed by deletion of helicases, PIF1 and MPH1, and by deletion of POL32, a subunit of DNA polymerase {delta}. All dna2{Delta} cells are temperature sensitive, have telomere length defects, and low levels of telomeric 3 single stranded DNA (ssDNA). Interestingly, Rfa1, a subunit of the major ssDNA binding protein RPA, and the telomere specific ssDNA binding protein Cdc13, often co-localize in dna2{Delta} cells. This suggests that telomeric defects often occur in dna2{Delta} cells. There are several plausible explanations for why the most critical function of Dna2 is at telomeres. Telomeres modulate the DNA damage response (DDR) at chromosome ends, inhibiting resection, ligation and cell cycle arrest. We suggest that Dna2 nuclease activity contributes to modulating the DNA damage response at telomeres by removing telomeric C-rich ssDNA and thus preventing checkpoint activation.

genetics↗

bioRxiv2017

Genome-scale genetic interactions position the Mitotic Exit Network as a major antagonist of transient Topoisomerase II deficiency.

Topoisomerase II (Top2) is the essential protein that resolves DNA catenations. When the Top2 is inactivated, mitotic catastrophe results from massive entanglement of chromosomes. Top2 is also the target of many first-line anticancer drugs, the so-called Top2 poisons. Often, tumours become resistant to these drugs by downregulating Top2. Here, we have compared two isogenic yeast strains carrying top2 thermosensitive alleles that differ in their resistance to Top2 poisons, the broadly-used poison-sensitive top2-4 and the poison-resistant top2-5. We found that top2-5 transits through anaphase faster than top2-4. In order to define the biological importance of this difference, we performed genome-scale Synthetic Gene Array (SGA) analyses during chronic sublethal Top2 downregulation and acute, yet transient, Top2 inactivation. We find that downregulation of cell cycle progression, especially the Mitotic Exit Network (MEN), protects against Top2 deficiency. In all conditions, genetic protection was stronger in top2-5, and this correlated with destabilization of anaphase bridges by execution of MEN. We suggest that mitotic exit may be a therapeutic target to hypersensitize cancer cells carrying downregulating mutations in TOP2.

genetics↗