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. 2023 Jan 10;51(2):796–805. doi: 10.1093/nar/gkac1263

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© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — Pre-steady-state analysis of LIG3β-catalyzed EJ in 10% (w/v) PEG. (A) Burst analysis of 1 and 2 nM LIG3β with 20 and 40 nM EJ substrates, respectively. Joined DNA represents the sum of both singly and doubly ligated products. (B) Representative DNA denaturing gel illustrating the formation and decay of the singly ligated product (labeled 2) followed by the formation of the doubly ligated product (labeled 1) during pre-steady-state blunt-end DNA ligation. Adenylylated DNA intermediate and unreacted blunt-end substrate are labeled 3 and 4, respectively. (C) Pre-steady-state quantification of singly and doubly ligated products for reactions of 20 nM LIG3β with 40 nM EJ substrates. Sum of singly and doubly ligated products is shown in blue. (D) Simplified mechanism for sequential ligation events by LIG3β. Experiments in (A) and (C) were performed in triplicate and data are presented as mean ± S.D.