Structures of Intermediates along the Catalytic Cycle of Terminal Deoxynucleotidyltransferase: Dynamical Aspects of the Two-Metal Ion Mechanism
| Autor: | Marc Delarue, Felix Romain, Jerome Gouge, Sandrine Rosario, Pierre Béguin |
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| Přispěvatelé: | Dynamique structurale des Macromolécules / Structural Dynamics of Macromolecules, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Biologie Moléculaire du Gène chez les Extrêmophiles (BMGE), Institut Pasteur [Paris], This work was supported by Association de la Recherche Contre le Cancer through grant 3155., We thank N. Expert-Bezançon and T. Efstratidis for their participation to the early stages of this project, especially the crystallization of the binary complex with the primer strand in malonate and the binary complex with dCTP and Zn2 +, respectively. We also thank D. Ptchelkine for help with site-directed mutagenesis of some Tdt mutants, A. Chaffotte for help in getting the visible absorption spectrum of Co2 + in solution and in the presence of concentrated Tdt, F. Schaeffer for complementary ITC experiments in the presence of Zn2 +, Dr Sun Un (Commissariat à l'Énergie Atomique et aux Énergies Alternatives) for electronic paramagnetic resonance experiments with Mn2 + and F. Rougeon (Institut Pasteur) for constant support and discussions. Finally, we thank the Proteopole platforms of A. Haouz and P. England and the staff of Soleil (Orsay) and European Synchrotron Radiation Facility (Grenoble) for help during data collection, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP) |
| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
Models
Molecular double-strand break DNA polymerase Protein Conformation MESH: Catalytic Domain Crystallography X-Ray Ligands MESH: Zinc DSB Substrate Specificity chemistry.chemical_compound MESH: Protein Conformation Structural Biology Catalytic Domain two-metal ion mechanism ssDNA MESH: Ligands A-DNA Polymerase 0303 health sciences biology 030302 biochemistry & molecular biology Cobalt isothermal titration calorimetry Zinc MESH: DNA Single-Stranded Metals polyethylene glycol single-strand DNA MESH: Models Molecular Protein Binding PDB MESH: Ions Stereochemistry DNA Single-Stranded MESH: DNA Nucleotidylexotransferase Catalysis 03 medical and health sciences DNA Nucleotidylexotransferase Protein Data Bank [CHIM.CRIS]Chemical Sciences/Cristallography Humans MESH: Protein Binding [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology Molecular Biology 030304 developmental biology Tdt X-ray crystallography Ions terminal deoxynucleotidyltransferase MESH: Humans MESH: Metals MD DNA translocation Active site ITC MESH: Catalysis MESH: Crystallography X-Ray PEG molecular dynamics A-site Crystallography MESH: Cobalt Catalytic cycle chemistry biology.protein MESH: Substrate Specificity Primer (molecular biology) structures of intermediates DNA |
| Zdroj: | Journal of Molecular Biology Journal of Molecular Biology, Elsevier, 2013, 425 (22), pp.4334-4352. ⟨10.1016/j.jmb.2013.07.009⟩ Journal of Molecular Biology, 2013, 425 (22), pp.4334-4352. ⟨10.1016/j.jmb.2013.07.009⟩ |
| ISSN: | 0022-2836 1089-8638 |
| DOI: | 10.1016/j.jmb.2013.07.009⟩ |
| Popis: | International audience; Terminal deoxynucleotidyltransferase (Tdt) is a non-templated eukaryotic DNA polymerase of the polX family that is responsible for the random addition of nucleotides at the V(D)J junctions of immunoglobulins and T-cell receptors. Here we describe a series of high-resolution X-ray structures that mimic the pre-catalytic state, the post-catalytic state and a competent state that can be transformed into the two other ones in crystallo via the addition of dAMPcPP and Zn(2+), respectively. We examined the effect of Mn(2+), Co(2+) and Zn(2+) because they all have a marked influence on the kinetics of the reaction. We demonstrate a dynamic role of divalent transition metal ions bound to site A: (i) Zn(2+) (or Co(2+)) in Metal A site changes coordination from octahedral to tetrahedral after the chemical step, which explains the known higher affinity of Tdt for the primer strand when these ions are present, and (ii) metal A has to leave to allow the translocation of the primer strand and to clear the active site, a typical feature for a ratchet-like mechanism. Except for Zn(2+), the sugar puckering of the primer strand 3' terminus changes from C2'-endo to C3'-endo during catalysis. In addition, our data are compatible with a scheme where metal A is the last component that binds to the active site to complete its productive assembly, as already inferred in human pol beta. The new structures have potential implications for modeling pol mu, a closely related polX implicated in the repair of DNA double-strand breaks, in a complex with a DNA synapsis. |
| Databáze: | OpenAIRE |
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