Fig. 6. Molecular basis of mutagenic ligation by LIG1^EE/AA.

a LIG1^EE/AA X-ray structure in complex with +1 nucleotide insertion DNA (insC4). The DBD (gray), AdD (teal), and OBD (gold) domains encircle a bulged nicked DNA substrate (bulged 3′-OH strand, magenta; 5′-P strand, blue; continuous strand, gray). b Structural overlay of LIG1^WT-DNA (PDB 6P09)¹⁴ and LIG1^EE/AA-bulged DNA complexes shows that the backbone path of the bulged DNA (magenta) is distorted at the HiFi Mg²⁺-binding pocket. LIG1^WT binds the HiFi Mg²⁺ (green) at the juncture between the 3′-OH of the upstream DNA (gold), AdD (teal), and DBD (gray). c Omit F_o–F_c electron density contoured at 2σ displayed for the bulged 3′-OH strand bound in the LIG1^EE/AA-bulged DNA complexes. d, Omit F_o–F_c electron density contoured at 2σ and displayed for the unbulged upstream 3′-OH strand bound in the LIG1^EE/AA-unbulged DNA complex. e–g Cartoon representations (top panels) and surface-filled representation of X-ray structures (bottom panels) depict protein–DNA contacts at the HiFi metal-binding sites of the LIG1^WT-DNA (e, PDB 6P09), LIG1^EE/AA-unbulged DNA (f), and LIG1^EE/AA-bulged insC4 DNA (g) complexes. The HiFi Mg²⁺ bridging the nt −3 and nt −4 nucleotides of the 3′-OH strand relative to the nick site. The AdD binds across the broken DNA strands with the nick positioned over the active site and makes contacts with nt +2 to nt −3 while the DBD contacts nt −4 to nt −6. f, Removal of the HiFi Mg²⁺ ligands (E592A/E346A) results in a cavity (EE/AA pocket). g The bulged extrahelical nucleotide (the fourth cytosine upstream of the DNA nick) in the LIG1^EE/AA-bulged DNA complex occupies the EE/AA pocket created by removal of the HiFi Mg²⁺-binding site. Source data for panels are provided as a Source data file.