PMC

(A) Structural alignment of LigI and LigIII shows the contrast between the high sequence conservation of the NTase and OBD domains and the low sequence conservation of the DBD domain. The secondary structure of LigIII, colored-coded by domain, is indicated above the amino acid sequence. Residues contacting DNA (green) or other domains (purple) are highlighted. The two helices that form a positively charged groove in unique to ligase III are highlighted in yellow, with key residues highlighted in bright yellow. (B) The domain structure of ligase III.

(A) Model of nick sensing by LigIII. The ZnF solution structure (PDB ID: 1uw0) was docked onto the DBD and DNA from the ΔZnF crystal structure to demonstrate how the ZnF-DBD module could bind to DNA in a nick-sensing mode. 73 residues of ligase III are missing from the two structures used to construct the model. This missing linker between the ZnF and NTase domains may interact with the positively charged groove in the DBD, as indicated by the black dots. (B) Unliganded ligase III adopts an extended conformation, as demonstrated by SAXS experiments. In the substrate recognition step, the ZnF is proposed to insert into the minor groove at the nick, recognizing flexibility in the DNA substrate. In the catalytic step, we hypothesize that the ZnF disengages to allow the DBD, NTase and OBD to fully encircle the nick, resembling in the ΔZnF crystal structure. (C) The two DNA binding regions of ligase III (the ZnF-DBD and NTase-OBD) could allow simultaneous binding of two DNAs to stimulate intermolecular ligations.

(A) Structural comparison of DBDs between ligase I (left) and ligase III reveals a unique positively charged groove of ligase III. (B) Close view of the positively charged groove, which is boxed in (A). (C) LigIIIβ, ΔZnF, and two mutants of LigIIIβ (K323E and R327E) with residue substitutions in the positively charged groove were assayed for blunt-end joining activity in a single turnover assay. 100 nM of proteins were reacted with 4 nM DNA substrates at 22 °C with different time points (10 sec, 20 sec, 40 sec, 1 min, 1.5 min, 2 min and 3 min). (D) The ligated fraction for each protein each protein in (C) [WT (●), ΔZnF (○), K323E (▼) and R327E (△)] is plotted. Error bars are standard deviations from three separate experiments. (E) The reciprocal charge-reversal mutants (K323E/E265K and R327E/D262R) and their parental single mutants were assayed for blunt-end and nick joining activity. 100 nM protein were reacted with DNA substrates at 22 °C for 1.5 min.

(A) Normalized pair distribution (P(r)) functions for the ΔZnF₇₅₅ protein in the presence and absence of DNA reveal a large conformational transition of the three conserved domains of LigIII (DBD-NTase-OBD) from an extended to a compact structure. (B) Comparisons of P(r) functions for LigIII₇₅₅ in the presence and absence of DNA show that ZnF-containing LigIII proteins are elongated even when bound to DNA, suggesting that the ZnF adopts flexible conformations during end joining. (C–F) Rigid body modeling of ΔZnF₇₅₅ (C), LigIII₇₅₅ (D), ΔZnF₇₅₅-DNA complex (E), and LigIII₇₅₅-DNA complex (F) by MD simulation and MES. For each protein complex, the top left panel shows a comparison of the experimental P(r) curve with those calculated from single best fit model (red) or the MES ensemble (green). In the ΔZnF₇₅₅-DNA complex (E), the crystal structure of LigIIIβ ΔZnF-DNA complex is used as the single model. In the top right panel, a comparison of D_max values for 10,000 models with their R_g values is shown. The best single model and the best fit group of MES conformers are indicated by red and green circles, respectively, with their relative volume fractions. The bottom panels show the single best fit model and three MES conformers with their relative volume fractions. The LigIII domains are colored as in .

(A) Ligase III bound to a nicked DNA substrate. The adenylated ΔZnF-Ligase IIIβ protein was captured in a pre-Step 2 complex with DNA prior to transfer of AMP to the DNA. The 5′ PO4 and 3′ ddC termini of the nicked DNA strand are highlighted in green and yellow, respectively. The crystallographic model contains 537 residues of the 697-residue protein fragment that was crystallized and 43 of the 44 nucleotides. The C-terminal 100 amino acids (residues 747–862) are disordered, indicating that this region is flexible. However, comparison to ligase homologues indicates that the entire OBD domain is observed. In addition, there are three disordered loops in the protein: two in the DNA-binding domain (DBD; residues 207–213 and 376–383) and one in the OB-fold domain (OBD; residues 666–691). All three of these loops are located on the exterior of the protein, away from the DNA and domain interfaces. (B) The surface view of ΔZnF-Ligase IIIβ bound to DNA shows that Ligase III forms a continuous ring about the DNA during Step 2 with the nicked DNA strand sequestered in the active site pocket of the NTase domain. (C) The DBD of Ligase III forms a platform for the highly distorted DNA, which is partially unwound and has an offset in the helical axis about the nick (left). The DBD interacts with the DNA through two helix-hairpin-helix motifs that insert into the minor groove on either side of the nick ().