DNA ligase I (LIG1) catalyzes the ligation of the nick repair intermediate after gap filling by DNA polymerase (pol) β during downstream steps of the base excision repair (BER) pathway. However, how LIG1 discriminates against the mutagenic 3′-mismatches incorporated by polβ at atomic resolution remains undefined. Here, we determine the X-ray structures of LIG1/nick DNA complexes with G:T and A:C mismatches and uncover the ligase strategies that favor or deter the ligation of base substitution errors. Our structures reveal that the LIG1 active site can accommodate a G:T mismatch in the wobble conformation, where an adenylate (AMP) is transferred to the 5′-phosphate of a nick (DNA-AMP), while it stays in the LIG1-AMP intermediate during the initial step of the ligation reaction in the presence of an A:C mismatch at the 3′-strand. Moreover, we show mutagenic ligation and aberrant nick sealing of dG:T and dA:C mismatches, respectively. Finally, we demonstrate that AP-endonuclease 1 (APE1), as a compensatory proofreading enzyme, removes the mismatched bases and interacts with LIG1 at the final BER steps. Our overall findings provide the features of accurate versus mutagenic outcomes coordinated by a multiprotein complex including polβ, LIG1, and APE1 to maintain efficient repair.

Ribonucleotides can be incorporated by DNA polymerases and the subsequent joining of 3'-OH and 5'-P ends in the phosphodiester backbone at the nick by DNA ligase during DNA replication and repair is critical for maintaining genome stability. Although it has been extensively studied for DNA polymerases across families, the sugar discrimination mechanism of a human DNA ligase at atomic resolution is entirely missing. Here, for the first time, we determine X-ray structure of DNA ligase I (LIG1) in complex with nick DNA containing rG:C at the 3'-end and capture the ligase at the final phosphodiester bond formation step of the ligation reaction involving an adenylate (AMP) release. Moreover, we show mutagenic end joining of the nick DNA substrate with preinserted 3'-rG:C by LIG1 in vitro. Our findings reveal an important source of ribonucleotides embedded in genomic DNA, which could come from the failure of LIG1 to discriminate against a ribonucleotide at the 3'-end during nick sealing step of DNA replication and repair.

ATP-dependent DNA ligases catalyze phosphodiester bond formation in the conserved three-step chemical reaction of nick sealing. Human DNA ligase I (LIG1) finalizes almost all DNA repair pathways following DNA polymerase-mediated nucleotide insertion. We previously reported that LIG1 discriminates mismatches depending on the architecture of the 3'-terminus at a nick, however the contribution of conserved active site residues to faithful ligation remains unknown. Here, we comprehensively dissect the nick DNA substrate specificity of LIG1 active site mutants carrying Ala(A) and Leu(L) substitutions at Phe(F)635 and Phe(F)F872 residues and show completely abolished ligation of nick DNA substrates with all 12 non-canonical mismatches. LIG1EE/AA structures of F635A and F872A mutants in complex with nick DNA containing A:C and G:T mismatches demonstrate the importance of DNA end rigidity, as well as uncover a shift in a flexible loop near 5'-end of the nick, which causes an increased barrier to adenylate transfer from LIG1 to the 5'-end of the nick. Furthermore, LIG1EE/AA/8oxoG:A structures of both mutants demonstrated that F635 and F872 play critical roles during steps 1 or 2 of the ligation reaction depending on the position of the active site residue near the DNA ends. Overall, our study contributes towards a better understanding of the substrate discrimination mechanism of LIG1 against mutagenic repair intermediates with mismatched or damaged ends and reveals the importance of conserved ligase active site residues to maintain ligation fidelity.

Ribonucleotides can be incorporated by DNA polymerases and the subsequent joining of 3'-OH and 5'-P ends in the phosphodiester backbone at the nick by DNA ligase during DNA replication and repair is critical for maintaining genome stability. Although it has been extensively studied for DNA polymerases across families, the sugar discrimination mechanism of a human DNA ligase at atomic resolution is entirely missing. Here, for the first time, we determine X-ray structure of DNA ligase I (LIG1) in complex with nick DNA containing rG:C at the 3'-end and capture the ligase at the final phosphodiester bond formation step of the ligation reaction involving an adenylate (AMP) release. Moreover, we show mutagenic end joining of the nick DNA substrate with preinserted 3'-rG:C by LIG1 in vitro. Our findings reveal an important source of ribonucleotides embedded in genomic DNA, which could come from the failure of LIG1 to discriminate against a ribonucleotide at the 3'-end during nick sealing step of DNA replication and repair.

ATP-dependent DNA ligases catalyze phosphodiester bond formation in the conserved three-step chemical reaction of nick sealing. Human DNA ligase I (LIG1) finalizes almost all DNA repair pathways following DNA polymerase-mediated nucleotide insertion. We previously reported that LIG1 discriminates mismatches depending on the architecture of the 3'-terminus at a nick, however the contribution of conserved active site residues to faithful ligation remains unknown. Here, we comprehensively dissect the nick DNA substrate specificity of LIG1 active site mutants carrying Ala(A) and Leu(L) substitutions at Phe(F)635 and Phe(F)F872 residues and show completely abolished ligation of nick DNA substrates with all 12 non-canonical mismatches. LIG1EE/AA structures of F635A and F872A mutants in complex with nick DNA containing A:C and G:T mismatches demonstrate the importance of DNA end rigidity, as well as uncover a shift in a flexible loop near 5'-end of the nick, which causes an increased barrier to adenylate transfer from LIG1 to the 5'-end of the nick. Furthermore, LIG1EE/AA/8oxoG:A structures of both mutants demonstrated that F635 and F872 play critical roles during steps 1 or 2 of the ligation reaction depending on the position of the active site residue near the DNA ends. Overall, our study contributes towards a better understanding of the substrate discrimination mechanism of LIG1 against mutagenic repair intermediates with mismatched or damaged ends and reveals the importance of conserved ligase active site residues to maintain ligation fidelity.