An expanded tool kit for the auxin‐inducible degron system in budding yeast
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TLDR
The construction of a series of vectors is reported that significantly enhance the versatility of this auxin‐inducible degron (AID) system in Saccharomyces cerevisiae and provides evidence for a general usefulness of the system.Abstract:
Fusion of inducible degradation signals, so-called degrons, to cellular proteins is an elegant method of controlling protein levels in vivo. Recently, a degron system relying on the plant hormone auxin has been described for use in yeast and vertebrate cells. We now report the construction of a series of vectors that significantly enhance the versatility of this auxin-inducible degron (AID) system in Saccharomyces cerevisiae. We have minimized the size of the degron and appended a series of additional epitope tags, allowing detection by commercial antibodies or fluorescence microscopy. The vectors are compatible with PCR-based genomic tagging strategies, allow for C- or N-terminal fusion of the degron, and provide a range of selection markers. Application to a series of yeast proteins, including essential replication factors, provides evidence for a general usefulness of the system.read more
Citations
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Reduced cellular levels of DNA polymerase delta alter replication dynamics and enzymology, and impair lagging-strand processing
TL;DR: It is proposed that replisomes with sub-stoichiometric Polδ are defective due to competition between nascent lagging-strand primers and terminating Okazaki fragments, and that this competition underlies both the replication and genome stability defects observed when cellular levels of Pol δ fall below a critical threshold.
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PICH promotes SUMOylated TopoisomeraseIIα dissociation from mitotic centromeres for proper chromosome segregation
Victoria A. Hassebroek,Hyewon Park,Nootan Pandey,Brooklyn T. Lerbakken,Vasilisa Aksenova,Alexei Arnaoutov,Mary Dasso,Yoshiaki Azuma +7 more
TL;DR: It is shown that PICH specifically promotes dissociation of SUMOylated TopoisomeraseIIα (TopoIIα) from mitotic chromosomes and that function leads to proper chromosome segregation.
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Dually targeted proteins regulate proximity between peroxisomes and partner organelles
Thorsten Stehlik,Elena Bittner,Jason Lam,Lazar Dimitrov,Isabelle Schöck,Jannik Harberding,Nikola Heymons,Maya Schuldiner,Einat Zalckvar,Michael Bölker,Randy Schekman,Johannes Freitag +11 more
TL;DR: It is proposed that dual targeting of proteins occurs at contact sites and that protein import per se contributes to the maintenance of these membrane proximities.
Posted ContentDOI
The Kinase Module of the Mediator regulates cell cycle re-entry and transcriptional response following DNA damage
TL;DR: In this paper , the authors performed an E-MAP analysis of Mec1 and Rad53, two essential kinases that coordinate the DNA damage response in budding yeast, and found that Rad53 and CKM physically interact in response to DNA damage.
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Conditional control of fluorescent protein degradation by an auxin-dependent nanobody
Katrin Daniel,Jaroslav Icha,Jaroslav Icha,Cindy Horenburg,Doris Müller,Caren Norden,Jörg Mansfeld +6 more
TL;DR: This work combines the advantages of auxin and nanobody-based degradation technologies creating an AID-nanobody to degrade GFP-tagged proteins at different cellular structures in a conditional and reversible manner in human cells.
References
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Journal ArticleDOI
Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae
Mark S. Longtine,Amos Mckenzie,Douglas J. Demarini,Nirav Shah,Achim Wach,Arndt Brachat,Peter Philippsen,John R. Pringle +7 more
TL;DR: A new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications that should further facilitate the rapid analysis of gene function in S. cerevisiae.
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TL;DR: The yeast Saccharomyces cerevisiae is now recognized as a model system representing a simple eukaryote whose genome can be easily manipulated and made particularly accessible to gene cloning and genetic engineering techniques.
Journal ArticleDOI
An auxin-based degron system for the rapid depletion of proteins in nonplant cells
TL;DR: The auxin-inducible degron (AID) system allowed rapid and reversible degradation of target proteins in response to auxin and enabled us to generate efficient conditional mutants of essential proteins in yeast as well as cell lines derived from chicken, mouse, hamster, monkey and human cells, thus offering a powerful tool to control protein expression and study protein function.
Journal ArticleDOI
Epitope tagging of yeast genes using a PCR‐based strategy: more tags and improved practical routines
Michael Knop,Katja Siegers,Gislene Pereira,Wolfgang Zachariae,Barbara Winsor,Kim Nasmyth,Elmar Schiebel +6 more
TL;DR: In the yeast Saccharomyces cerevisiae, molecular biological techniques have been developed that use a simple PCR‐based strategy to introduce epitope tags to chromosomal loci to produce PCR amplificable modules.
Journal ArticleDOI
Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation
Philipp Stelter,Helle D. Ulrich +1 more
TL;DR: These findings assign a function to SUMO during S phase and demonstrate how ubiquitin and SUMO, by regulating the accuracy of replication and repair, contribute to overall genomic stability.