title: Structural basis for a novel mechanism of DNA bridging and alignment in eukaryotic DSB DNA repair.
abstract: Eukaryotic DNA polymerase mu of the PolX family can promote the association of the two 3'-protruding ends of a DNA double-strand break (DSB) being repaired (DNA synapsis) even in the absence of the core non-homologous end-joining (NHEJ) machinery. Here, we show that terminal deoxynucleotidyltransferase (TdT), a closely related PolX involved in V(D)J recombination, has the same property. We solved its crystal structure with an annealed DNA synapsis containing one micro-homology (MH) base pair and one nascent base pair. This structure reveals how the N-terminal domain and Loop 1 of Tdt cooperate for bridging the two DNA ends, providing a templating base in trans and limiting the MH search region to only two base pairs. A network of ordered water molecules is proposed to assist the incorporation of any nucleotide independently of the in trans templating base. These data are consistent with a recent model that explains the statistics of sequences synthesized in vivo by Tdt based solely on this dinucleotide step. Site-directed mutagenesis and functional tests suggest that this structural model is also valid for Pol mu during NHEJ.
authors.affiliation: Unité de Dynamique Structurale des Macromolécules, Institut Pasteur, UMR 3528 du C.N.R.S., Paris, France.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Gouge
authors.affiliation: Unité de Dynamique Structurale des Macromolécules, Institut Pasteur, UMR 3528 du C.N.R.S., Paris, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Rosario
authors.affiliation: Unité de Dynamique Structurale des Macromolécules, Institut Pasteur, UMR 3528 du C.N.R.S., Paris, France.
authors.forename: Félix
authors.initials: F
authors.lastname: Romain
authors.affiliation: Institut de Physique Théorique, CEA-Saclay, CNRS URA 2306, Gif-sur-Yvette, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Poitevin
authors.affiliation: Unité de Biologie Moléculaire du Gène chez les Extrêmophiles, Institut Pasteur, Paris, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Béguin
authors.affiliation: Unité de Dynamique Structurale des Macromolécules, Institut Pasteur, UMR 3528 du C.N.R.S., Paris, France marc.delarue@pasteur.fr.
authors.forename: Marc
authors.initials: M
authors.lastname: Delarue
mesh_terms: D000818:Animals; D001483:Base Sequence; D018360:Crystallography, X-Ray; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D004253:DNA Nucleotidylexotransferase / Q000737:chemistry / Q000502:physiology; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000502:physiology; D005057:Eukaryotic Cells / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D011487:Protein Conformation; D060152:V(D)J Recombination
chemical_list: C407743:DNA polymerase mu; D004253:DNA Nucleotidylexotransferase; D004259:DNA-Directed DNA Polymerase
keywords: DNA repair; DNA synapsis; X‐ray crystallography; micro‐homology base pair; non‐homologous end‐joining