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. 2013 Feb 1;41(5):3162–3172. doi: 10.1093/nar/gkt043

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© The Author(s) 2013. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — The influence of Cdc6 mutants on pre-RC formation. Pre-RC assembly was performed as described in the methods section. A 30% load of the pre-RC proteins is shown in lanes 1–6. The use of the mutants is indicated in the figures. The gel was silver stained to visualize the proteins; however, the smallest subunit of the Orc1–6 complex stains only weakly. (A) Cdc6, Cdc6 K114E (Walker A) and Cdc6 R332E (sensor-2) were used in pre-RC assays in the presence of ATP (lanes 8–16) or ATPγS (lane 7). Pre-RCs were washed with a low salt buffer (lanes 7–9, 11, 12, 14 and 15) or a high salt buffer (lanes 10, 13 and 16). (B) Cdc6, Cdc6 E224G (Walker B) and Cdc6 N263A (sensor-1) were used in pre-RC assays in the presence of ATP (lanes 8–16) or ATPγS (lane 7). Pre-RCs were washed with a low salt buffer (lanes 7–9, 11, 12, 14 and 15) or a high salt buffer (lanes 10, 13 and 16).