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. 2011 Apr 20;39(14):6137–6147. doi: 10.1093/nar/gkr220

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© The Author(s) 2011. 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/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — ϕC31 Int is inactivated by pre-incubation with EDTA. Int (1 µM) was pre-incubated with increasing concentrations of EDTA prior to the addition of substrates (pRT600 and pRT702) to assay integration (top panel, lanes 1–6). At the same time as adding substrates metal salts were added [final concentration 0.5 mM: NiSO₄ (top panel, lanes 7–12), MgSO₄, (top panel, lanes 13–18), CaSO₄ (middle panel, lanes 1–6), FeSO₄ (middle panel, lanes 7–11), ZnSO₄ (middle panels, lanes 13–18), MnSO₄ (bottom panel, lanes 1–6), CuSO₄ (bottom panel, lanes 7–12) or CoSO₄ (bottom panel, lanes 13–18)]. Recombination reactions were incubated (30°C, 1 h) and recombinant products were detected by restriction with HindIII and separation by agarose gel electrophoresis. The substrates pRT702 (2491 bp) or pRT600 (3035 bp) are each cut once by HindIII and the product is a co-integrate (pRT600702) that is cleaved by HindIII to yield 5435 and 91 bp bands. M are molecular weight markers (NEB, 1 kb ladder). Only ZnSO₄ could restore full integration activity.