Ribonucleolytic resection is required for repair of strand displaced nonhomologous end-joining intermediates

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Abstract

Nonhomologous end-joining (NHEJ) pathways repair DNA double-strand breaks (DSBs) in eukaryotes and many prokaryotes, although it is not reported to operate in the third domain of life, archaea. Here, we describe a complete NHEJ complex, consisting of DNA ligase (Lig), polymerase (Pol), phosphoesterase (PE), and Ku from a mesophillic archaeon, Methanocella paludicola (Mpa). Mpa Lig has limited DNA nick-sealing activity but is efficient in ligating nicks containing a 3' ribonucleotide. Mpa Pol preferentially incorporates nucleoside triphosphates onto a DNA primer strand, filling DNA gaps in annealed breaks. Mpa PE sequentially removes 3' phosphates and ribonucleotides from primer strands, leaving a ligatable terminal 3' monoribonucleotide. These proteins, together with the DNA end-binding protein Ku, form a functional NHEJ break-repair apparatus that is highly homologous to the bacterial complex. Although the major roles of Pol and Lig in break repair have been reported, PE's function in NHEJ has remained obscure. We establish that PE is required for ribonucleolytic resection of RNA intermediates at annealed DSBs. Polymerase-catalyzed strand-displacement synthesis on DNA gaps can result in the formation of nonligatable NHEJ intermediates. The function of PE in NHEJ repair is to detect and remove inappropriately incorporated ribonucleotides or phosphates from 3' ends of annealed DSBs to configure the termini for ligation. Thus, PE prevents the accumulation of abortive genotoxic DNA intermediates arising from strand displacement synthesis that otherwise would be refractory to repair.

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