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. 2015 Jan 5;43(2):973–986. doi: 10.1093/nar/gku1364

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© The Author(s) 2015. 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/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. — D161A replacement in loop L1 allows RecA to use double-stranded DNA as a cofactor for LexA repressor cleavage. (A, B, C) DNA cofactors for LexA cleavage. LexA (4.7 μM) was incubated for the indicated times with 1.0-μM RecA-wt (A) or recA-D161A (B) in the presence of the indicated DNA (18 μM) and 5.0-mM MgCl₂. (D, E) Effects of MgCl₂ concentrations on LexA cleavage. LexA was incubated for 5 or 20 min (as indicated) with 1.0-μM RecA-wt or recA-D161A in the presence of 18-μM single-stranded DNA (D) or double-stranded DNA (E), in reaction mixtures containing the indicated concentrations of MgCl₂. DNA added to the reaction mixture: ss, single-stranded DNA; FI, negatively supercoiled closed circular DNA (form I DNA); FIII, linear double-stranded DNA (form III DNA). Proteins in signals were indicated on the left side of the panels: BSA, bovine serum albumin; CK, creatine phosphokinase from the ATP-regenerating system; RecA, RecA-wt or recA-D161A; LexA, uncleaved LexA; C and N, C-terminal and N-terminal fragments, respectively, of cleaved LexA. Lane M: protein markers, with sizes in kDa indicated on the left sides of the gels in (B) and (E); lane D, recA-D161A; lane W, RecA-wt.