Figure 7

Footprinting of the bottom strands of the MJcj and MJpc substrates. Footprinting reactions were run on an 8% polyacrylamide sequencing gel together with the unbound DNA reactions (FR) and the G+A Maxam-Gilbert sequencing reactions (G+A). Vertical grey and black rectangular bars represent weakly and strongly protected residues respectively. The bands in the G+A and footprinted lanes are identified with dots and/or short horizontal lines. The DNA sequence of the bottom strand of the MCJ is shown to the left of the G+A lanes and is numbered as R1 to R39 and L1 to L37 reading from the abutted ends towards to the inside ends of the two termini. The spacer base (G) of the MCJ is numbered as 0. (A) Two hour exposure of the gel. (B) Overnight exposure of the gel facilitated the ready distinction of weak and tight binding. Bars labeled (a) identify sequences in the CD of IRL that are disengaged in the nicked (MJpc) substrate and more tightly bound in the covalently closed (MJcj) substrate. Bars labeled (b) in the CD of IRR and the PBD of IRL, indicate sequences that are more strongly protected in MJpc than in MJcj. The bars labeled (c) at the terminal trinucleotide of IRR identify differences in binding affinity to this sequence of the two substrates. The (d) labels indicate the loss of binding affinity to the PBD of IRR in the cleaved substrate compared to the covalently joined substrate bringing the protection pattern of the former more in line with that of the single IRR end (see Figure 9). CD: cleavage domain; IRR/IRL: right and left inverted repeats; MJcj: covalently joined minicircle junction substrate; MJpc: precleaved minicircle junction substrate; PBD: protein binding domain.