Structural evidence for an in trans base selection mechanism involving Loop1 in polymerase μ at an NHEJ double-strand break junction
| Autor: | Marc Delarue, Sandrine Rosario, Michael R. Lieber, Jérôme Loc’h, Christina A. Gerodimos, Mustafa Tekpinar |
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| Přispěvatelé: | Institut Pasteur [Paris], University of Southern California (USC), Dynamique structurale des Macromolécules / Structural Dynamics of Macromolecules, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), This work was supported by the 'Fondation ARC pour la recherche sur la cancer' through a post-doctoral fellowship to J.L. MT is supported by the PAUSE program (Collège de France, IP). Work in the lab of MRL is supported by NIH, Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS) |
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
DNA End-Joining Repair DNA polymerase DNA repair DNA damage Stereochemistry Recombinant Fusion Proteins Eukaryotic DNA replication DNA-Directed DNA Polymerase DNA and Chromosomes Biochemistry Substrate Specificity 03 medical and health sciences chemistry.chemical_compound Mice Isomerism DNA Nucleotidylexotransferase Catalytic Domain [CHIM.CRIS]Chemical Sciences/Cristallography structural biology Animals DNA Breaks Double-Stranded Amino Acid Sequence Molecular Biology Polymerase X-ray crystallography 030102 biochemistry & molecular biology biology [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry Molecular Biology/Structural Biology [q-bio.BM] V(D)J recombination Cell Biology DNA Protein Structure Tertiary 030104 developmental biology Terminal deoxynucleotidyl transferase chemistry biology.protein Sequence Alignment non-homologous DNA end joining |
| Zdroj: | J Biol Chem Journal of Biological Chemistry Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2019, pp.jbc.RA119.008739. ⟨10.1074/jbc.RA119.008739⟩ Journal of Biological Chemistry, 2019, pp.jbc.RA119.008739. ⟨10.1074/jbc.RA119.008739⟩ |
| ISSN: | 0021-9258 1083-351X |
| DOI: | 10.1074/jbc.RA119.008739⟩ |
| Popis: | International audience; Eukaryotic DNA polymerase (Pol) X family members such as Pol μ and terminal deoxynucleotidyl transferase (TdT) are important components for the nonhomologous DNA end-joining (NHEJ) pathway. TdT participates in a specialized version of NHEJ, V(D)J recombination. It has primarily non-templated polymerase activity, but can take instructions across strands from the downstream dsDNA, and both activities are highly dependent on a structural element called Loop1. However, it is unclear whether Pol μ follows the same mechanism because the structure of its Loop1 is disordered in available structures. Here, we used a chimeric TdT harboring Loop1 of Pol μ that recapitulated the functional properties of Pol μ in ligation experiments. We solved three crystal structures of this TdT chimera bound to several DNA substrates at 1.96-2.55 Å resolutions, including a full DNA-double strand break (DSB) synapsis. We then modeled the full Pol μ sequence in the context of one these complexes. The atomic structure of an NHEJ junction with a pol X construct that mimics Pol μ in a reconstituted system explained the distinctive properties of Pol μ compared with TdT. The structure suggested a mechanism of base selection relying on Loop1 and taking instructions via the in trans templating base independently of the primer strand. We conclude that our atomic-level structural observations represent a paradigm shift for the mechanism of base selection in the polX family of DNA polymerases. |
| Databáze: | OpenAIRE |
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