Mechanistic Basis for a Single Amino Acid Residue Mutation Causing Human DNA Ligase 1 Deficiency, A Rare Pediatric Disease

Abstract

In mammalian cells, DNA ligase 1 (LIG1) functions as the primary DNA ligase in both genomic replication and single-strand break repair. Several reported mutations in human LIG1, including R305Q, R641L, and R771W, cause LIG1 syndrome, a primary immunodeficiency. While the R641L and R771W mutations, respectively located in the nucleotidyl transferase and oligonucleotide binding domains, have been biochemically characterized and shown to reduce catalytic efficiency, the recently reported R305Q mutation within the DNA binding domain (DBD) remains mechanistically unexplored. The R641L and R771W mutations are known to decrease the catalytic activity of LIG1 by affecting both interdomain interactions and DNA binding during catalysis, without significantly impacting overall DNA affinity. To elucidate the molecular basis of the LIG1 syndrome-causing R305Q mutation, we purified this single-residue mutant protein and investigated its secondary structure, protein stability, DNA binding affinity, and catalytic efficiency. Our findings reveal that the R305Q mutation significantly impairs the function of LIG1 by disrupting the DBD-DNA interactions, leading to a 7–21-fold lower DNA binding affinity and a 33–300-fold reduced catalytic efficiency of LIG1. Additionally, the R305Q mutation slightly decreases LIG1’s protein stability by 2 to 3.6 °C, on par with the effect observed previously with either the R641L or R771W mutant. Collectively, our results uncover a new mechanism whereby the R305Q mutation impairs LIG1-catalyzed nicked DNA ligation, resulting in LIG1 syndrome, and highlight the crucial roles of the DBD-DNA interactions in tight DNA binding and efficient LIG1 catalysis.