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

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 non-templated 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 polX family of DNA polymerases.
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Submitted on : Monday, July 1, 2019 - 4:08:26 PM
Last modification on : Monday, January 13, 2020 - 5:08:18 PM

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Jerome Loc'H, Christina Gerodimos, Sandrine Rosario, Mustafa Tekpinar, Michael Lieber, et al.. Structural evidence for an in trans base selection mechanism involving Loop1 in Polymerase mu at an NHEJ double-strand break junction. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2019, pp.jbc.RA119.008739. ⟨10.1074/jbc.RA119.008739⟩. ⟨pasteur-02170007⟩

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