Molecular basis for RNA discrimination by human DNA ligase 1

Abstract

DNA ligase 1 (LIG1) finalizes DNA replication and repair by catalyzing the joining of DNA nicks. LIG1 is highly specific for DNA-DNA junctions over DNA-RNA junctions, discriminating strongly against a single ribonucleotide at the 5' side of the nick. This selectivity of LIG1 prevents futile and potentially mutagenic DNA-RNA cleavage and re-ligation cycles during Okazaki fragment maturation or ribonucleotide excision repair of genome-embedded ribonucleotide monophosphates (rNMPs), but the determinants of LIG1 rNMP discrimination are ill-defined. We report structural and kinetic analysis of LIG1 DNA-RNA complexes showing that LIG1 employs an aromatic steric gate to stabilize the enzyme-substrate complex and directly exclude rNMP-containing polynucleotides. Mutation of this RNA gate compromises the adenylyl-transfer and nick-sealing reactions but decreases the discrimination against an rNMP-containing substrate by ∼3600-fold. Our results establish the role of the conserved steric gate in ribonucleotide discrimination by high-fidelity (HiFi) DNA ligases at each step of the ligation reaction, which has parallels to the ribonucleotide discrimination by HiFi DNA polymerases.

Graphical Abstract

Figure 1.

High-fidelity (HiFi) ligation by LIG1 prevents a futile cycle of resealing DNA–RNA junctions. (A) The three-step ATP-dependent ligation reaction. (B) Futile cycle of ligation and cleavage of RNA-containing DNA. (C) The multi-domain LIG1 is comprised of a flexible N-terminal domain (dark gray) containing a nuclear localization signal and a PIP box motif (orange) that mediates interactions with Proliferating cell nuclear antigen (PCNA) during DNA replication and a catalytic core (residues 232–919) formed by the DNA-binding domain (DBD, teal), the adenylylation domain (AdD, pink), and the OB-fold domain (OBD, gray). Crystal structure of LIG1·Mg²⁺·AMP–DNA complex (PDB6P09) showing the proximity of the F872 residue (green) to the 5′-adenylylated DNA. The core domains encircle the DNA substrate (yellow). The 5′-phosphate of the nick is adenylylated (orange), and the catalytic and HiFi Mg²⁺ ions (red) are bound in the active site and HiFi metal binding site, respectively.

Figure 2.

Kinetic characterization of LIG1 WT on DNA and RDNA substrates. (A) A representative denaturing gel of steady-state ligation reactions of WT LIG1 with the R28mer and 28mer at 1 or 20 mM Mg²⁺. Single-turnover ligation of DNA and RDNA substrates by LIG1 WT. Single-turnover ligation assays contained 800 nM enzyme and 80 nM of either the 28mer DNA (B) or the RDNA (C) substrate (filled circles represent ligation reaction products; open circles represent adenylylated intermediates). Reactions were performed in the absence of ATP and in the presence of 1 mM Mg²⁺. Reaction traces were fit using a two-step irreversible model in Berkeley Madonna and the best-fit values are provided in Supplementary Table S4. (D) Steady-state substrate dependences at 1 mM free Mg²⁺ demonstrate that WT LIG1 exhibits a significantly slower k_cat with the R28mer substrate containing a single ribonucleotide at the nick in 5′ position. (E) Plot showing sugar selectivity of LIG1 WT at 1 mM free Mg²⁺. The discrimination for k_seal and k_transfer is the ratio of k^DNA/k^RDNA. The discrimination for k_cat/K_M (ligated) is defined as  to account for the fraction of abortive ligation that is observed for the RDNA substrate.

Figure 3.

Effects of the F872 mutations on DNA and RDNA dependences. (A) F872 packs tightly against the 5′-terminal deoxyribose sugar. The presence of a ribonucleotide at this position would cause a steric clash between the F872 side chain and the 2′-OH from the ribose. Conserved ribo-gate loop residues (R871, F872, P873, and R874) stabilize the ends of the DNA nick by participating in molecular contacts with the DNA strand and active site residues. (B) Sequence alignment of LIG1 homologs, eukaryotic ligases, ATP-dependent bacterial DNA ligase (Pm LIG), and chlorella virus DNA ligase (ChV LIG) generated by Clustal Omega showing stringently conserved F872 amongst eukaryotic, archaeal, and bacterial ATP-dependent DNA ligases as well as chlorella virus. (C) A representative denaturing gel of steady-state ligation reactions of F872A and F872L with the R28mer or 28mer at 1 or 20 mM Mg²⁺. (D) Plot showing the amount of abortive ligation by WT LIG1 and mutants observed in the steady-state R28mer dependences in the presence of 1 mM Mg²⁺ (left) and 20 mM Mg²⁺ (right). (E) Plot showing the relative k_cat/K_M for the DNA substrate compared to the RDNA substrate [ ]. All values are reported as the mean ± standard deviation of N ≥ 3 replicates.

Figure 4.

Structural basis of ribonucleotide discrimination by LIG1. Omit Fo–Fc electron density contoured at 1.5σ displayed for the RNA-gate loop residues (R871, F872, P873, and R874) engaging the 5′-adenylylated nick in the WT–DNA complex (PDB6P0C) (A), F872A–DNA complex (B), F872A–RDNA complex (C), and F872L–DNA complex (D). Ca-DNA backbone and protein side chain–5′-terminal sugar moiety distances in the WT–DNA complex (PDB6P0C) (E), F872A–DNA complex (F), F872A–RDNA complex (G), and F872L–DNA complex (H). Surface representation displayed for complementary contacts between the adenylylated 5′-terminal nucleotide and F872 in the WT–DNA complex (PDB6P0C) (I), A872 in the F872A–DNA complex (J), A872 in the F872A–RDNA complex (K), and L872 in the F872L–DNA complex (L). (M) F872 (gray) participates in sugar–π stacking interactions with the deoxyribose moiety of the 5′-terminal nucleotide at the nick. F872A (blue) mutation creates free space between the alanine and the 5′-deoxyribose sugar, leading to the loss of the critical complementary protein–sugar interface (N). Normally, WT LIG1 uses F872 as an RNA gate to block the C2′-OH of the 5′-terminal ribonucleotide. Superimposition of the 5′-terminal nucleotides of the WT–DNA complex (gray) and F872A–RDNA complex (orange) shows that the C2′-OH of the 5′-terminal ribonucleotide causes steric clash with the phenylalanine side chain. However, F872A mutant can accommodate and form a stable complex with the RDNA substrate. (O) Superimposition of the 5′-terminal nucleotide of F872L–DNA (teal) and F872A–RDNA (orange) complexes shows how the C2′-OH of a 5′-terminal ribonucleotide would cause steric clash with the leucine side chain.

Figure 5.

LIG1 utilizes distinct 3′ and 5′ fidelity mechanisms to ensure faithful ligation. LIG1 utilizes an aromatic RNA gate (F872) in ribonucleotide discrimination to avoid ligation of DNA nick containing 5′ RNA, which is distinct from the mechanism for discrimination against upstream mismatches and single nucleotide insertions by a HiFi divalent metal (Mg²⁺)-dependent 3′ DNA substrate binding mode.