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

Abstract

Nonhomologous end-joining (NHEJ) pathways repair DNA doublestrand 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. D NA double-strand breaks (DSBs) are one of the most lethal forms of damage encountered by cells (1). Nonhomologous end joining (NHEJ) is the primary pathway for repairing DSBs in higher eukaryotes (2). NHEJ does not require the presence of a sister chromatid, in contrast to homologous recombination, and therefore can operate in quiescent cells. The direct repair and ligation of DSBs by NHEJ is considered to be more error prone because breaks are mended without the assistance of an intact DNA template.The higher eukaryotic NHEJ complex is composed primarily of the ligase IV, X-ray repair cross-complementing protein 4 (XRCC4), and XRCC4-like factor (XLF) complex (the LXX complex), DNA-dependent protein kinase, catalytic subunit (DNA-PKcs), and Ku70/80 (1, 3). Ku and DNA-PKcs both assist in bridging the gap between the broken termini by promoting end synapsis. NHEJ repair of nonhomologous breaks requires remodeling of the DNA termini by processing enzymes, including polymerases (Pol λ and μ) and nucleases [Artemis and flap endonuclease 1 (Fen-1)], to prepare them for ligation (4-7). The LXX complex is recruited by Ku, which enables the ligation of the DNA ends.Most bacterial organisms also possess an NHEJ complex, composed of a more limited set of proteins, including ligase D (LigD) and Ku (8,9), which is required for DSB repair in stationary phase (10). The mycobacterial LigD gene encodes a multifunctional enzyme which, along with Ku, is ...

NHEJ enzymes LigD and Ku participate in stationary-phase mutagenesis in Pseudomonas putida

“…In gapped substrates, POL domain has the ability to dislocate and realign template, extending the primer by inserting nucleotides complementary to template bases distal to the primer terminus [30,34]. In the case of break termini containing 3 overhangs that lack a primer strand, LigD POL can facilitate the formation of functional primer-template substrate by annealing the 3 overhanging strands from opposing breaks to form a gapped intermediate that can be extended in trans [31,33,35]. Hence, LigD POL may cause various insertions, deletions and base substitutions during the processing of DSBs.…”

“…LigD proteins often consist of an ATP-dependent DNA ligase domain (LIG), a polymerase domain (PolDom or POL), and a 3 -phosphoesterase domain (PE), called also as a nuclease domain, that could account for the DNA ends processing, gap filling and sealing steps in NHEJ [15,16,18,23,[25][26][27][28][29][30][31][32][33]. POL domain catalyses either non-templated single nucleotide additions to a blunt-ended duplex DNA (primase activity) or possesses DNA-dependent DNA/RNA gap filling polymerase activities [18,[23][24][25]27].…”

Efficient processing of abasic sites by bacterial nonhomologous end-joining Ku proteins

“…Recently, the presence of a bacterial related NHEJ complex has been reported also in archaea and constituted by Ku, and stand-alone ligase, polymerase and phosphoesterase proteins (20). …”

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

“…Therefore, we examined whether NHEJ could be established in Methanosarcina for use in conjunction with the Cas9-sgRNA complex. For this purpose, we chose the NHEJ machinery from the closely related methanogen M. paludicola, which has previously been reconstituted in vitro (23,24). An artificial operon encoding four M. paludicola NHEJ proteins [DNA ligase (Lig), polymerase (Pol), phosphoesterase (PE), and Ku] was synthesized and transcriptionally fused to the moderately expressed serC promoter (25) to allow transcription in M. acetivorans (Fig.…”