Ribonucleolytic resection is required for repair of strand displaced NHEJ intermediates

Non homologous end-joining (NHEJ) pathways repair DNA double-strand breaks (DSBs) in eukaryotes and many prokaryotes, although 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 ligase has limited DNA nick sealing activity but is efficient at ligating nicks containing a 3ʼ ribonucleotide. Mpa Pol preferentially incorporates NTPs onto a DNA primer strand, filling DNA gaps in annealed breaks. Mpa PE sequentially removes 3ʼ-phosphates and ribonucleotides from primer strands, leaving a ligatable terminal 3ʼ-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, PEʼs 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 non-ligatable NHEJ intermediates. PEʼs function in NHEJ repair is to detect and remove inappropriately incorporated ribonucleotides, or phosphates, from 3ʼ 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 would otherwise be refractory to repair. 3 NHEJ is a major DNA break repair pathway in eukaryotes and prokaryotes but assumed to be absent in archaea. This study establishes that a functionally homologous pathway is present in archaea. We have reconstituted archaeal NHEJ repair in vitro, demonstrating that it is closely related to the bacterial apparatus and preferentially repairs breaks using RNA intermediates. We identify a role for a functionally unascribed nuclease in preventing the accumulation of genotoxic repair intermediates produced by strand displacement. This study has important implications for our understanding of the mechanisms of DNA break repair by NHEJ and the evolution of end-joining pathways.