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EMBO J. 2015 Apr 15;34(8):1126-42. doi: 10.15252/embj.201489643. Epub 2015 Mar 11.

Structural basis for a novel mechanism of DNA bridging and alignment in eukaryotic DSB DNA repair.

Author information

1
Unité de Dynamique Structurale des Macromolécules, Institut Pasteur, UMR 3528 du C.N.R.S., Paris, France.
2
Institut de Physique Théorique, CEA-Saclay, CNRS URA 2306, Gif-sur-Yvette, France.
3
Unité de Biologie Moléculaire du Gène chez les Extrêmophiles, Institut Pasteur, Paris, France.
4
Unité de Dynamique Structurale des Macromolécules, Institut Pasteur, UMR 3528 du C.N.R.S., Paris, France marc.delarue@pasteur.fr.

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.

KEYWORDS:

DNA repair; DNA synapsis; X‐ray crystallography; micro‐homology base pair; non‐homologous end‐joining

PMID:
25762590
PMCID:
PMC4406656
DOI:
10.15252/embj.201489643
[Indexed for MEDLINE]
Free PMC Article

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