Molecular basis for DNA strand displacement by NHEJ repair polymerases

Abstract

The non-homologous end-joining (NHEJ) pathway repairs DNA double-strand breaks (DSBs) in all domains of life. Archaea and bacteria utilize a conserved set of multifunctional proteins in a pathway termed Archaeo-Prokaryotic (AP) NHEJ that facilitates DSB repair. Archaeal NHEJ polymerases (Pol) are capable of strand displacement synthesis, whilst filling DNA gaps or partially annealed DNA ends, which can give rise to unligatable intermediates. However, an associated NHEJ phosphoesterase (PE) resects these products to ensure that efficient ligation occurs. Here, we describe the crystal structures of these archaeal (Methanocella paludicola) NHEJ nuclease and polymerase enzymes, demonstrating their strict structural conservation with their bacterial NHEJ counterparts. Structural analysis, in conjunction with biochemical studies, has uncovered the molecular basis for DNA strand displacement synthesis in AP-NHEJ, revealing the mechanisms that enable Pol and PE to displace annealed bases to facilitate their respective roles in DSB repair.

Figure 1.

Crystal structure of an archaeal NHEJ phosphodiesterase. (A) A ribbon diagram representation of the crystal structure of Mpa PE. The hydrophobic core of the enzyme is comprised of a β-barrel containing 9 anti-parallel β-strands. A vanadate ion (grey) is bound in the active site adjacent to a 3₁₀ helix. A bound magnesium ion is also indicated by a green sphere. (B, C) Surface representations of the Mpa PE crystal structure. The active site is denoted by the vanadate and magnesium ions, and the loop that encloses the active site is shown in a ‘closed’ position. (D) A ribbon diagram of the 1.79 Å resolution structure of Mpa PE with detail of the active site. H40, H46 and D48 are involved in coordinating a catalytic metal ion represented here by magnesium, whilst R50, H82 and Y86 are involved in coordinating the vanadate ion, which likely occupies the position of the scissile phosphate in phosphoesterase reactions. The side chains are covered by electron density maps (2F_o-F_c), shown in blue mesh with a 1.5-σ cutoff.

Figure 2.

Critical function and conservation of active site residues in the AP–NHEJ phosphoesterase. (A) This amino acid alignment shows the three residues that coordinate the metal ion in the catalytic site (red box), whilst the three residues that coordinate the scissile phosphate are shown (green box). All six residues are strictly conserved in all species listed here. The conserved glutamate that is predicted to be essential for 3′-phosphatase activity is also shown (purple box). Aligned species; Mpa—Methanocella paludicola (archaea), Dly—Dehalogenimonas lykanthroporepellens (bacteria), Mba—Methanosarcina barkeri (archaea), Cko—Candidatus korarchaeum cryptofilim (archaea), Dac—Desulfobacca acetoxidans (bacteria), Smo—Streptomyces monomycini (bacteria), Mna—Marinobacter nanhaiticus (bacteria), Smu—Salipiger mucosus (bacteria), Mtu—Mycobacterium tuberculosis (bacteria), Pae—Pseudomonas aeruginosa (bacteria), Rco—Ricinus Communis (plantae). (B) A scheme showing the reaction mechanism for Mpa PE, detailing our nomenclature for the reaction products. n-R-R is a primer of indeterminate length that contains two successive ribonucleosides at the 3′ end. n-R-P is the intermediate reaction product following the removal of a ribonucleoside that still retains a 3′-phosphate. n-R-OH is the final reaction product that has had the 3′-phosphate moiety removed and terminates in a hydroxyl group. (C), (D) Phosphoesterase reactions contained 300 nM Mpa PE and 300 nM Mpa PE H82A mutant where indicated. Reactions also contained 30 nM 5′-fluorescein labelled substrate and 5mM Mn. The two 3′ RNA bases of substrate in (D) are non-complementary with the template strand, and create a 3′ flap. The wild type reactions were incubated for 30, 60 and 90 min, and the H82A mutants were incubated for 90 min, at 37°C.

Figure 3.

Structural and functional conservation of AP–NHEJ polymerases. (A) A ribbon representation of the crystal structure Mpa Pol. Loop 1 is highlighted in dark blue, whilst Loop 2 is highlighted in cyan. (B) A ribbon representation of the Mpa Pol crystal structure manually docked with an incoming nucleotide and DNA substrate derived from the Mtu PolDom structures PDB:3PKY and PDB:4MKY, respectively (15,36). (C) A comparison of loop positioning between Mpa Pol (magenta) and Mtu PolDom (cream). Loop 1 and Loop 2 are shown in dark blue and cyan, respectively. The loops occupy very similar positions, despite differing in amino acid composition, suggesting that the function of the loops is conserved. The Mtu PolDom figure was generated using the polymerase structure, PDB 2IRU (20).

Figure 4.

Structural features of NHEJ polymerases involved in displacement synthesis. (A) Gap filling extensions reactions contained 300 nM Mtu PolDom or Mpa Pol and 30 nM 5′-fluorescein labelled substrate. Reactions also contained 62.5 μM of either ATP, CTP, or a mixture of all 4 NTPs (mix), and 5 mM Mn and were incubated for the indicated time periods. (B) A ribbon representation of the crystal structure of Mtu PolDom engaging a 1 nucleotide gap substrate with an incoming nucleotide, generated with the previously published Mtu PolDom structures PDB:3PKY and PDB:4MKY (Brissett et al., 2011, 2013). The phenyalanines, arginine and proline residues that contact the downstream dsDNA interface are shown in stick format (dark blue). (C) A surface representation of the Mtu PolDom model with gap DNA substrate. The structural wedge that meets the downstream dsDNA is clearly visible in this format. The DNA substrate components are labelled as primer, template and D-strand (downstream strand). (D) A detailed view of dsDNA interface with Mtu PolDom and the potential amino acid residues involved in displacement synthesis, shown in ribbon and stick format.

Figure 5.

A comparison of dislocation activities of residues potentially involved in displacement synthesis. (A) A schematic of the gapped DNA substrates used for the gap filling and displacement synthesis reactions, including details of the templating bases in the gap. (B-E) DNA extension assays with Mtu PolDom wt, K16A, F63A and F64A, respectively. Reactions contained 300 nM AP–NHEJ polymerase with 30 nM 5′-fluorescein labelled substrate and 5mM Mn and were incubated for 1 h at 37ºC. Reactions contained either a mix of NTPs or individual NTPs (ATP, CTP, GTP or UTP) as indicated.

Figure 6.

R53 is directly involved in splaying the template/D-strand junction of DNA to allow displacement synthesis to occur. (A) A DNA extension assay containing 300 nM Mtu PolDom^R53A, with 30 nM 5′-fluorescein labelled substrate and 5mM Mn. Reactions contained either a mix of NTPs or individual ATP, CTP, GTP or UTP, as indicated, and reactions were incubated for 1 h at 37ºC. (B) The binding contacts made by R53 of Mtu PolDom with neighbouring residues and the nucleotides at the ss/ds DNA interface (C) The conserved phosphate binding pocket is highlighted in orange, the strictly conserved arginine implicated in displacement synthesis is highlighted in purple, the DNA splaying phenylalanine residues are shown in green, and the conserved active site residues are shown in red. These key regions of the AP–NHEJ are conserved across bacteria, archaea and even plants. Aligned species; Mpa—Methanocella paludicola (archaea), Smo—Streptomyces monomycini (bacteria), Pae—Pseudomonas aeruginosa (bacteria), Rco—Ricinus Communis (plantae), Mtu—Mycobacterium tuberculosis (bacteria).

Figure 7.

Potential roles for displacement synthesis during NHEJ repair. A schematic diagram demonstrating how NHEJ polymerases (green) may utilise displacement synthesis in order to temporarily stabilise DSB intermediates. NHEJ Pols can ingress into fully annealed blunt DNA ends or overhanging ends. The arginine-tipped wedge in the polymerase allows the annealed DNA to be ‘opened’ to expose regions of microhomology. This MMEJ process enables the break termini to be synapsed back together, forming more stable intermediates that can be further processed and repaired more efficiently and precisely. This displacement synthesis mechanism can be likened to the joining of the ends of a broken zip, with the polymerase acting as the slider that adds teeth as it moves across the break to gain traction with the other side. The new teeth displace the ones that were already zipped, allowing the two broken ends to become more stably connected. After this connection has been made, the synapsed break can then be rejoined back together.