Sequence-specific DNA binding activity of the cross-brace zinc finger motif of the piggyBac transposase

Autor: Nadine Assrir, Nathalie Mathy, Silke Andrea Wieninger, Michael Nilges, Fred Dyda, Julien Bischerour, Eric Guittet, Ewen Lescop, Xianghong Li, Benjamin Bardiaux, Séverine Moriau, Alison B. Hickman, Nancy L. Craig, Jennifer L Taylor, Mireille Bétermier, Nelly Morellet
Přispěvatelé: Institut de Chimie des Substances Naturelles (ICSN), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Howard Hughes Medical Institute (HHMI), Johns Hopkins University (JHU), Bioinformatique structurale - Structural Bioinformatics, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), National Institutes of Health [Bethesda] (NIH), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Intramural Research Program of the NIH and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (to J.L.T., A.B.H., F.D.), The Centre National de la Recherche Scientifique (CNRS) and the Agence Nationale de la Recherche (ANR) [ANR-14-CE10-0005-01 (PIGGYPACK) to N.M., E.L., N.A., E.G., N. Mathy, J.B., M.B.], The Institut de Chimie des Substances Naturelles (ICSN) (to S.M), European Union [FP7-IDEAS-ERC 294809 to M.N., Howard Hughes Medical Institute, Department of Molecular Biology & Genetics, Johns Hopkins University School of Medicine (to X.L. N.L.C.). Funding for open access charge: Laboratoire de Biologie et Chimie Structurales, Institut de Chimie des Substances Naturelles, CNRS UPR 2301, Université Paris-Saclay, 91198 Gif sur Yvette cedex, France., ANR-14-CE10-0005,PIGGYPACK,Domestication de transposases et épigénétique: Impact sur la dynamique des génomes(2014), European Project: 294809,EC:FP7:ERC,ERC-2011-ADG_20110310,BAYCELLS(2012), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Rok vydání: 2018
Předmět:
Zdroj: Nucleic Acids Research
Nucleic Acids Research, Oxford University Press, 2018, 46 (5), pp.2660-2677. ⟨10.1093/nar/gky044⟩
Nucleic Acids Research, 2018, 46 (5), pp.2660-2677. ⟨10.1093/nar/gky044⟩
PubMed Central
ISSN: 1362-4962
0305-1048
Popis: International audience; The piggyBac transposase (PB) is distinguished by its activity and utility in genome engineering, especially in humans where it has highly promising therapeutic potential. Little is known, however, about the structure-function relationships of the different domains of PB. Here, we demonstrate in vitro and in vivo that its C-terminal Cysteine-Rich Domain (CRD) is essential for DNA breakage, joining and transposition and that it binds to specific DNA sequences in the left and right transposon ends, and to an additional unexpectedly internal site at the left end. Using NMR, we show that the CRD adopts the specific fold of the cross-brace zinc finger protein family. We determine the interaction interfaces between the CRD and its target, the 5'-TGCGT-3'/3'-ACGCA-5' motifs found in the left, left internal and right transposon ends, and use NMR results to propose docking models for the complex, which are consistent with our site-directed mutagenesis data. Our results provide support for a model of the PB/DNA interactions in the context of the transpososome, which will be useful for the rational design of PB mutants with increased activity.
Databáze: OpenAIRE
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