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. Author manuscript; available in PMC: 2009 May 2.
Published in final edited form as: J Mol Biol. 2008 Mar 4;378(3):653–665. doi: 10.1016/j.jmb.2008.02.058

Figure 6. The constrained ABAB heterotetramer distinguishes between different arrangements of LoxP and LoxM7 13 bp repeats.

Figure 6

(a)–(d) Complex structures and electrophoretic analysis of assembly competence and turnover rate. The relevant complexes or substrate pairs are diagrammed on the left. Example phosphorimages are shown of complex assembly titrations (left gel panel) and time course experiments (right gel panel). Titration reactions were carried out as described in Figure 4 and MATERIALS AND METHODS. Complex concentrations in nanomolar are given above the lanes. For time course reactions (1200 nM complex), aliquots were removed at the indicated times, given in seconds, quenched, and electrophoresed. The 220 bp fragment is indicated by the colored dot. The reactions are equimolar CreAA+ALSHG-F with (a) LoxM7P x LoxM7P; (b) LoxP x LoxM7; and (c) LoxM7P x LoxPM7. Similar data for the control reaction of the unconstrained heterotetramer CreWT+CreALSHG in a LoxPM7 x LoxM7P cross are given in (d).

(e) Quantification of titration and time course data. The concentration- (upper panel) or time-dependences (lower panel and inset) of substrate turnover are given for each protein-DNA combination in (a)–(d): CreAA+ALSHG-F/LoxM7P x LoxM7P (filled diamonds, blue line); CreAA+ALSHG-F/LoxPM7 x LoxM7P (filled circles, cyan line); CreAA+ALSHG-F/LoxP x LoxM7 (filled squares, magenta line); and CreWT+ALSHG/LoxPM7 x LoxM7P (open triangles, red line). Average, normalized data points and their standard deviations are indicated, except for CreAA+ALSHG-F/LoxPM7 x LoxM7P, which turned over less than 3% substrate at 1200 nM complex. The curves colors correspond to the reactions indicated by dots at the right of (a)–(d). Complex assembly titration curves were derived from averaged Hill parameters from 2–5 experiments, given in Table 1. The time course progress curves were calculated from a biphasic kinetic model31 using averaged parameters from 2–4 experiments, given in Table 1. The inset shows the expanded time course from 0 to 300 seconds.