Molecular Basis for DNA Double-Strand Break Annealing and Primer Extension by an NHEJ DNA Polymerase

Abstract

SummaryNonhomologous end-joining (NHEJ) is one of the major DNA double-strand break (DSB) repair pathways. The mechanisms by which breaks are competently brought together and extended during NHEJ is poorly understood. As polymerases extend DNA in a 5′-3′ direction by nucleotide addition to a primer, it is unclear how NHEJ polymerases fill in break termini containing 3′ overhangs that lack a primer strand. Here, we describe, at the molecular level, how prokaryotic NHEJ polymerases configure a primer-template substrate by annealing the 3′ overhanging strands from opposing breaks, forming a gapped intermediate that can be extended in trans. We identify structural elements that facilitate docking of the 3′ ends in the active sites of adjacent polymerases and reveal how the termini act as primers for extension of the annealed break, thus explaining how such DSBs are extended in trans. This study clarifies how polymerases couple break-synapsis to catalysis, providing a molecular mechanism to explain how primer extension is achieved on DNA breaks.

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].…”

TruePrime is a novel method for whole-genome amplification from single cells based on TthPrimPol

“…1). Moreover, the significant amino acid sequence similarity with pRN1 PrimPol and also with the polymerization domain (PolDom) of Mycobacterium tuberculosis LigD (not shown), whose three-dimensional (3D) structures have been solved264344, was sufficient to generate a 3D model for Tth PrimPol in complex with DNA and nucleotide substrates (Fig. 1b; see Methods for details).…”

Structural Determinants Responsible for the Preferential Insertion of Ribonucleotides by Bacterial NHEJ PolDom

“…The inspection of the ternary complex of Mt-PolDom shows the presence of a lysine residue (Lys66) that packs against the template nucleotide, maintaining its spatial orientation [22][23][24]; see also Figure 1. This lysine residue is absolutely conserved in all LigDs [ [24], see also Figure 1B] and would correspond to Pa-LigD Lys606.…”

“…Whereas, in most cases, LigD is a multifunctional protein where the ligase domain (LigDom) is fused to a phosphoesterase (PEDom) and/or a polymerization (PolDom) domain responsible for processing incompatible termini, in several bacteria those domains exist as stand-alone proteins (reviewed in [21]). Extensive biochemical and structural characterization of the PolDom of Mycobacterium tuberculosis LigD (Mt-PolDom) allowed to decipher how NHEJ operates in bacteria to repair DSBs containing 3 -protruding ends [22][23][24]. Initially, Ku binds to both sides of the DSB and recruits LigD whose PolDom recognizes specifically the recessive 5 -P termini, forming a preternary precatalytic complex with Mn 2+ ions and the incoming nucleotide that forms a Watson-Crick base pair with the templating nucleotide nearest the 5 -P end [23].…”