title: Structural Basis for a New Templated Activity by Terminal Deoxynucleotidyl Transferase: Implications for V(D)J Recombination.
abstract: Eukaryotic DNA polymerase of the polX family, such as pol μ and terminal deoxynucleotidyl transferase (TdT), are key components of the non-homologous end-joining or V(D)J recombination machinery, respectively. The established role of TdT is to add random nucleotides during V(D)J recombination. Here we show that TdT also has a templated-polymerase activity, similar to pol μ, in the presence of higher concentrations of a downstream DNA duplex, and performs a micro-homology single base-pair search to align the DNA synapsis. To understand the molecular basis of this alignment, we solve crystal structures of TdT with four DNA strands and study the influence of the 3' protruding end. Two mutations in TdT inspired by sequence alignments with pol μ further improve the templated activity. We propose that both templated and untemplated activities of TdT are needed to explain the distributions of lengths of N regions observed experimentally in T cell receptors and antibodies.
authors: Unité de Dynamique Structurale des Macromolécules, Institut Pasteur, UMR 3528 du C.N.R.S., 25 rue du Dr Roux, 75015 Paris, France.
authors: Jérôme
authors: J
authors: Loc'h
authors: Unité de Dynamique Structurale des Macromolécules, Institut Pasteur, UMR 3528 du C.N.R.S., 25 rue du Dr Roux, 75015 Paris, France.
authors: Sandrine
authors: S
authors: Rosario
authors: Unité de Dynamique Structurale des Macromolécules, Institut Pasteur, UMR 3528 du C.N.R.S., 25 rue du Dr Roux, 75015 Paris, France. Electronic address: marc.delarue@pasteur.fr.
authors: Marc
authors: M
authors: Delarue
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004253:DNA Nucleotidylexotransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D060152:V(D)J Recombination
chemical_list: D009711:Nucleotides; D011994:Recombinant Proteins; D004247:DNA; C407743:DNA polymerase mu; D004253:DNA Nucleotidylexotransferase; D004259:DNA-Directed DNA Polymerase
keywords: V(D)J recombination; X-ray crystallography; double-strand-break DNA repair; micro-homology base pair