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. Author manuscript; available in PMC: 2011 Jul 27.
Published in final edited form as: Biochemistry. 2010 Jul 27;49(29):6165–6176. doi: 10.1021/bi100503w

Figure 3. A unique positively charged groove in the DBD is important for intermolecular DNA ligation.

Figure 3

(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.