Figure 7

Excision Time-course analysis at 30°C: circular versus linear. (a) Diagrammatic representation of excision products obtained (after MOS1 activity) from an Nco I-linearized pBC-3Tet3: double strand DNA cleavages at one ITR yield two fragments (α and β) or (γ and δ). Double-stranded DNA cleavages at each ITR end yield three fragments (β, δ and 3Tet3). (b) Analysis of cleavage products obtained from super coiled pBC-3Tet3 DNA substrate. Reactions were performed from 0 to 24 hours at 30°C, using 80 nM MOS1. After deproteinizing, products were Nco I digested and loaded onto agarose gel (left panel). Molecular weights (in kbp) are shown on the left. The different DNA species observed are indicated on the right, using the same codification as in (a). After quantification (ImageGauge V4.22 software), DNA substrate and α + δ bands (in fmoles) were plotted as a function of time (right panel, Prism Software). Averages and standard errors were calculated from five independent replicates, using Prism software. T1/2 is the time required producing 50% of α + δ (in fmoles, indicated by an arrow). (c) Analysis of cleavage products obtained from a linear pBC-3Tet3 DNA substrate. Reactions were performed and analyzed as in (b). The only difference was that cleavage products were directly loaded onto agarose gel. (d) After quantification (ImageGauge V4.22 software), β + γ bands obtained in (b: circular donor) and (c: linear donor) were plotted (in fmoles) as a function of time (right panel, Prism Software). Averages and standard errors were calculated from five independent replicates (Prism software).