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 0 -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.

Translesion and Repair DNA Polymerases: Diverse Structure and Mechanism

“…Recently reported structures of TdT in complex with two DNA fragments reveal how DNA ends are connected without any complementary base pair (139). A 3´-primer end of a DNA fragment and an incoming dNTP can be aligned in the active site of TdT for the synthesis reaction when a mismatched template DNA is provided in trans (Fig.…”

Non‐homologous end joining: Common interaction sites and exchange of multiple factors in the DNA repair process

“…These polymerases have slightly different characteristics and varying fidelities: all can employ a template‐dependent mechanism whereby the polymerase can bridge small regions of complementarity (1–2 bp) for gap filling; in addition Polμ and TdT have template‐independent terminal transferase activity which adds nucleotides to the ends of DNA. Non‐templated nucleotide additions and the low fidelity of the PolX polymerases contribute to the generation of junctional diversity during the antigen receptor development by VDJ recombination .…”

Structural evidence for an in trans base selection mechanism involving Loop1 in polymerase μ at an NHEJ double-strand break junction

“…Note that in the TdT-Pol mu chimera 29 amino acids were changed, containing Loop1 (20 residues). In addition, 4 residues upstream and 5 residues downstream were changed, thereby also including the SD1 region which stands out as "maximally different" between TdT sequences and Pol mu sequences (25). Comparable template-dependent polymerase activity was observed in TdT-μ chimera and Pol μ using an in cis DNA substrate ( Figure 1A), whereas wild-type TdT displays an essentially un-templated polymerase activity in the same conditions (22).…”

“…It interacts with residues in the fingers domain (L260), the palm domain (Q379, R403 and D434), and the thumb domain (R454, R461, D473, N474 and H475) of the protein (Figure 6B-D). The interactions with the incoming base involve only main chain atoms of Loop1 ( Figure 6C) and the templating base interacts with the conserved residue R461, whose mutation into an alanine has a strong deleterious effect in TdT (25). Importantly, all of the interactions of Loop1 in the chimera construct with the rest of the TdTlike structure involve residues that are conserved in Pol μ or subject to a conservative substitution ( Figure 6C).…”

“…-The side chains of Q393B and T397 make hydrogen bonds with the DNH motif (D473, N474 and H475), also called SD2 region (22) or SD2 motif (25), or thumb mini-loop motif (16), localized in the β8-β9 loop ( Figure 6D). Mutations of this motif in human pol µ (16) resulted in loss of function.…”