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. 2015 Apr 22;290(23):14618–14625. doi: 10.1074/jbc.M115.641787

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© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 3. — Activity of PC assembled from intact and pre-nicked substrates. A, scheme of PC formation using intact (PC) or pre-nicked (PC-nick) 12/23RSS substrates (open and filled triangles) in Ca²⁺. Subsequent DNA cleavage in the presence of Mg²⁺/Mn²⁺ led to the production of SEC. B, PC formation by simultaneous mixing of all components. Superdex-200 elution profiles of protein alone, PC, and PC-nick preparation were superimposed. Arrows indicate the elution points of molecular mass markers (443 and 150 kDa). C and D, DNA cleavage in PC assembled from intact (C) and pre-nicked 12/23RSS (D) in the presence of MgCl₂ and MnCl₂ for 1–2 h. The ³²P labels on DNA strands are marked with asterisks. DNA cleavage products were separated on a TBE-urea gel. E, analysis of the metal ion concentrations, reaction time, and molar ratios of RAGs to DNA substrates in paired cleavage of pre-nicked RSSs by RAG1/M2. F, graphic summary of DNA cleavage reactions shown in E.