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

J Biol Chem. 2019 Jul 5;294(27):10579-10595. doi: 10.1074/jbc.RA119.008739. Epub 2019 May 28.


Eukaryotic DNA polymerase (Pol) X family members such as Pol μ and terminal deoxynucleotidyl transferase (TdT) are important components for the nonhomologous DNA end-joining (NHEJ) pathway. TdT participates in a specialized version of NHEJ, V(D)J recombination. It has primarily nontemplated polymerase activity but can take instructions across strands from the downstream dsDNA, and both activities are highly dependent on a structural element called Loop1. However, it is unclear whether Pol μ follows the same mechanism, because the structure of its Loop1 is disordered in available structures. Here, we used a chimeric TdT harboring Loop1 of Pol μ that recapitulated the functional properties of Pol μ in ligation experiments. We solved three crystal structures of this TdT chimera bound to several DNA substrates at 1.96-2.55 Å resolutions, including a full DNA double-strand break (DSB) synapsis. We then modeled the full Pol μ sequence in the context of one these complexes. The atomic structure of an NHEJ junction with a Pol X construct that mimics Pol μ in a reconstituted system explained the distinctive properties of Pol μ compared with TdT. The structure suggested a mechanism of base selection relying on Loop1 and taking instructions via the in trans templating base independently of the primer strand. We conclude that our atomic-level structural observations represent a paradigm shift for the mechanism of base selection in the Pol X family of DNA polymerases.

Keywords: DNA bridging; DNA damage; DNA polymerase; DNA polymerase Pol X family; DNA repair; DNA synapsis; Pol μ catalytic cycle; V(D)J recombination; X-ray crystallography; double-strand break; junctional diversity; non-homologous DNA end joining; structural biology; ternary complex.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Catalytic Domain / genetics
  • DNA / chemistry
  • DNA / metabolism
  • DNA Breaks, Double-Stranded
  • DNA End-Joining Repair*
  • DNA Nucleotidylexotransferase / chemistry*
  • DNA Nucleotidylexotransferase / genetics
  • DNA Nucleotidylexotransferase / metabolism
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism
  • Isomerism
  • Mice
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Sequence Alignment
  • Substrate Specificity


  • Recombinant Fusion Proteins
  • DNA
  • DNA Nucleotidylexotransferase
  • DNA-Directed DNA Polymerase
  • Polm protein, mouse

Associated data

  • PDB/1JMS
  • PDB/1XSN
  • PDB/2IHM
  • PDB/5D46