LAUSR

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.