6 results on '"Lebbink, Joyce"'
Search Results
2. The miniature CRISPR-Cas12m effector binds DNA to block transcription.
- Author
-
Wu, Wen Y., Mohanraju, Prarthana, Liao, Chunyu, Adiego-Pérez, Belén, Creutzburg, Sjoerd C.A., Makarova, Kira S., Keessen, Karlijn, Lindeboom, Timon A., Khan, Tahseen S., Prinsen, Stijn, Joosten, Rob, Yan, Winston X., Migur, Anzhela, Laffeber, Charlie, Scott, David A., Lebbink, Joyce H.G., Koonin, Eugene V., Beisel, Chase L., and van der Oost, John
- Subjects
- *
DNA , *COMPLEMENTARY DNA , *CYTIDINE deaminase , *CRISPRS , *CATALYTIC domains - Abstract
CRISPR-Cas are prokaryotic adaptive immune systems. Cas nucleases generally use CRISPR-derived RNA guides to specifically bind and cleave DNA or RNA targets. Here, we describe the experimental characterization of a bacterial CRISPR effector protein Cas12m representing subtype V-M. Despite being less than half the size of Cas12a, Cas12m catalyzes auto-processing of a crRNA guide, recognizes a 5′-TTN′ protospacer-adjacent motif (PAM), and stably binds a guide-complementary double-stranded DNA (dsDNA). Cas12m has a RuvC domain with a non-canonical catalytic site and accordingly is incapable of guide-dependent cleavage of target nucleic acids. Despite lacking target cleavage activity, the high binding affinity of Cas12m to dsDNA targets allows for interference as demonstrated by its ability to protect bacteria against invading plasmids through silencing invader transcription and/or replication. Based on these molecular features, we repurposed Cas12m by fusing it to a cytidine deaminase that resulted in base editing within a distinct window. [Display omitted] • Type V-M is an additional subtype of type V CRISPR-Cas system (Cas12-U1) • The miniature Cas12m lacks target-specific as well as collateral nuclease activity • Guide-specific silencing by Cas12m occurs through dsDNA binding • A synthetic Cas12m-cytidine deaminase has a distinct C-to-T base editing window In this work, Wu et al. describe the characterization of a small CRISPR effector protein Cas12m representing subtype V-M. Cas12m is guided by a single crRNA to stably bind but not cleave complementary double-stranded DNA, potentially contributing to host defense by silencing the expression of invader DNA. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
3. Isolation of Functional Tubulin Dimers and of Tubulin-Associated Proteins from Mammalian Cells.
- Author
-
Yu, Nuo, Signorile, Luca, Basu, Sreya, Ottema, Sophie, Lebbink, Joyce H.G., Leslie, Kris, Smal, Ihor, Dekkers, Dick, Demmers, Jeroen, and Galjart, Niels
- Subjects
- *
TUBULINS , *CYTOSKELETON , *DIMERS , *CELL migration , *BRAIN abnormalities , *FLUORESCENT proteins , *PROTEIN-protein interactions - Abstract
Summary The microtubule (MT) cytoskeleton forms a dynamic filamentous network that is essential for many processes, including mitosis, cell polarity and shape, neurite outgrowth and migration, and ciliogenesis [ 1, 2 ]. MTs are built up of α/β-tubulin heterodimers, and their dynamic behavior is in part regulated by tubulin-associated proteins (TAPs). Here we describe a novel system to study mammalian tubulins and TAPs. We co-expressed equimolar amounts of triple-tagged α-tubulin and β-tubulin using a 2A “self-cleaving” peptide and isolated functional fluorescent tubulin dimers from transfected HEK293T cells with a rapid two-step approach. We also produced two mutant tubulins that cause brain malformations in tubulinopathy patients [ 3 ]. We then applied a paired mass-spectrometry-based method to identify tubulin-binding proteins in HEK293T cells and describe both novel and known TAPs. We find that CKAP5 and the CLASPs, which are MT plus-end-tracking proteins with TOG(L)-domains [ 4 ], bind tubulin efficiently, as does the Golgi-associated protein GCC185, which interacts with the CLASPs [ 5 ]. The N-terminal TOGL domain of CLASP1 contributes to tubulin binding and allows CLASP1 to function as an autonomous MT-growth-promoting factor. Interestingly, mutant tubulins bind less well to a number of TAPs, including CLASPs and GCC185, and incorporate less efficiently into cellular MTs. Moreover, expression of these mutants in cells impairs several MT-growth-related processes involving TAPs. Thus, stable tubulin-TAP interactions regulate MT nucleation and growth in cells. Combined, our results provide a resource for investigating tubulin interactions and functions and widen the spectrum of tubulin-related disease mechanisms. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
4. Mismatch Repair Inhibits Homeologous Recombination via Coordinated Directional Unwinding of Trapped DNA Structures.
- Author
-
Tham, Khek-Chian, Hermans, Nicolaas, Winterwerp, Herrie?H.K., Cox, Michael?M., Wyman, Claire, Kanaar, Roland, and Lebbink, Joyce?H.G.
- Subjects
- *
DNA structure , *RECOMBINANT DNA , *DNA repair , *ESCHERICHIA coli , *BACTERIAL DNA , *BACTERIAL genomes - Abstract
Summary: Homeologous recombination between divergent DNA sequences is inhibited by DNA mismatch repair. In Escherichia coli, MutS and MutL respond to DNA mismatches within recombination intermediates and prevent strand exchange via an unknown mechanism. Here, using purified proteins and DNA substrates, we find that in addition to mismatches within the heteroduplex region, secondary structures within the displaced single-stranded DNA formed during branch migration within the recombination intermediate are involved in the inhibition. We present a model that explains how higher-order complex formation of MutS, MutL, and DNA blocks branch migration by preventing rotation of the DNA strands within the recombination intermediate. Furthermore, we find that the helicase UvrD is recruited to directionally resolve these trapped intermediates toward DNA substrates. Thus, our results explain on a mechanistic level how the coordinated action between MutS, MutL, and UvrD prevents homeologous recombination and maintains genome stability. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
5. HSF2BP Interacts with a Conserved Domain of BRCA2 and Is Required for Mouse Spermatogenesis.
- Author
-
Brandsma I, Sato K, van Rossum-Fikkert SE, van Vliet N, Sleddens E, Reuter M, Odijk H, van den Tempel N, Dekkers DHW, Bezstarosti K, Demmers JAA, Maas A, Lebbink J, Wyman C, Essers J, van Gent DC, Baarends WM, Knipscheer P, Kanaar R, and Zelensky AN
- Subjects
- Animals, BRCA2 Protein genetics, Carrier Proteins genetics, Cell Line, Tumor, Heat-Shock Proteins genetics, Humans, Mice, Mutation, Missense, Protein Domains, BRCA2 Protein metabolism, Carrier Proteins metabolism, Heat-Shock Proteins metabolism, Spermatogenesis
- Abstract
The tumor suppressor BRCA2 is essential for homologous recombination (HR), replication fork stability, and DNA interstrand crosslink repair in vertebrates. We identify HSF2BP, a protein previously described as testis specific and not characterized functionally, as an interactor of BRCA2 in mouse embryonic stem cells, where the 2 proteins form a constitutive complex. HSF2BP is transcribed in all cultured human cancer cell lines tested and elevated in some tumor samples. Inactivation of the mouse Hsf2bp gene results in male infertility due to a severe HR defect during spermatogenesis. The BRCA2-HSF2BP interaction is highly evolutionarily conserved and maps to armadillo repeats in HSF2BP and a 68-amino acid region between the BRC repeats and the DNA binding domain of human BRCA2 (Gly2270-Thr2337) encoded by exons 12 and 13. This region of BRCA2 does not harbor known cancer-associated missense mutations and may be involved in the reproductive rather than the tumor-suppressing function of BRCA2., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
6. Variations on the ABC.
- Author
-
Lebbink JH and Sixma TK
- Subjects
- Models, Molecular, Protein Conformation, Adenosine Triphosphatases chemistry
- Published
- 2005
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.