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. 2017 Mar 28;45(10):5863–5876. doi: 10.1093/nar/gkx209

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© The Author(s) 2017. 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 (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 2. — Uracil excision assays with single stranded and double stranded substrates. Assays were carried out at 37°C for 30 min with 1 μg of BdiUng (∼32 pmol monomer) or EcoUng (∼4 pmol) with (+) or without (–) pre-incubation with 1 μg of Ugi (∼100 pmol) as described in Materials and Methods using ∼0.1 pmol of ssU9 (A) or GU9 (B) DNAs, and resolved on 8 M urea PAGE (15%). (C) Electrophoretic mobility shift assay to analyze complex formation of BdiUng with Ugi. After incubation of enzyme with/without Ugi, reaction products were resolved on 15% native PAGE (29:1 crosslinking, pH 6.8). While complex EcoUng-Ugi complex formed is found to migrate adjacent to pure complex control, no band indicative of BdiUng-Ugi complex could be seen. (D) Uracil excision assay on ssU9 with 2 μg of B. diazoefficiens cell free extracts with/without pre-incubation with varying amounts of Ugi. E. coli TG1 cell free extract (2 μg) was used as a control. Reaction products were resolved on 8 M urea PAGE (15%).