Abstract
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.
Highlights
Eukaryotic DNA polymerase (Pol) X family members such as Pol and terminal deoxynucleotidyl transferase (TdT) are important components for the nonhomologous DNA end-joining (NHEJ) pathway
The expression of TdT is limited to primary lymphoid organs, where B- and T-cell maturation occurs, and TdT does not participate in the NHEJ pathway in other cell types [12]
We demonstrate the biological relevance of this Tdt- chimera protein using an in vitro NHEJ ligation assay, which shows functional properties similar to Pol
Summary
Eukaryotic DNA polymerase (Pol) X family members such as Pol 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 Loop. Crystal structures of TdT in the presence of a full DNA synapsis could be obtained [15] and showed that Loop is crucial to maintain a tight binding of TdT across the DNA synapsis (Fig. 1C). This raised the question of whether Loop has the same role in Pol and possibly uses a similar mechanism of base selection. We present the structure of a ternary complex of TdT- chimera with a full DNA–DSB synapsis and an incoming nucleotide, which could be used as a model for the Pol –DNA DSB complex
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