Mechanistic Investigation of the DNA Ligase I Deficiency Variant A624T

Mammalian cells rely on DNA ligase 1 (LIG1) as the key ligase for DNA replication and single-strand break repair. LIG1 syndrome, a primary immunodeficiency, arises from various missense mutations in LIG1, such as R305Q, R641L, R771W, and the recently identified A624T. The R641L and R771W variants, positioned within the nucleotidyltransferase (NTD) and oligonucleotide-binding domains, respectively, reduce catalytic efficiency by perturbing interdomain interactions and catalysis-related DNA binding, despite preserving overall DNA affinity. The R305Q mutation in the DNA binding domain (DBD) severely impairs LIG1 function by disrupting DBD-DNA interactions and the major steps of the DNA ligation mechanism. However, the molecular basis of dysfunction for the A624T variant (located in the NTD) has remained poorly defined. To elucidate the molecular basis underlying this pathogenic variant, we investigated the effects of A624T on protein stability, DNA-binding affinity, and pre-steady-state ligation kinetics. We found that the A624T mutation causes a 3-fold reduction in the rate-limiting adenylyl transfer step at saturating Mg²⁺ and a 34-fold reduction at lower Mg²⁺ concentrations while having insignificant effects on protein thermostability, DNA-binding affinity, or the nick-sealing rate. This biochemical profile resembles that of R641L and R771W but is clearly distinct from the severely impaired R305Q variant. Molecular modeling suggests that the A624T substitution subtly perturbs the active site by displacing the catalytic Mg²⁺ ion and weakening the R641-D600 salt bridge. Collectively, our results indicate that A624T causes modest alterations to the LIG1 active site and ligation kinetics, providing mechanistic insight into how this variant contributes to LIG1 syndrome.