Figure 1 Biochemical analyses of PaqCI cleavage activity

Panel a: PaqCI demonstrates substrate turnover when acting on a stoichiometric excess of DNA targets. Digest time points are quenched at 10 minutes and 60 minutes with protein concentration ranging from 0.3-8.0 units with the DNA concentration held at 200 nM in each vial. In the 10 minute digest, complete cleavage of the oligo is only seen in concentrations from 1.0-8.0 units, with only partial cleavage to no cleavage in the rest of the lower concentrations. At 60 minutes, almost all concentrations exhibit complete cleavage of the oligo, with bands fading at 0.12 units and below. One unit of enzyme corresponds to 24 ng of enzyme (the amount required to digest 1 microgram of lambda DNA to completion in 1 hour at 37°C in a 50 microliter volume); the range of enzyme concentrations shown corresponds to 140 nM (16 units in 50 microliter reaction volume) to 2.2 nM (0.25 units in 50 microliter reaction volume). Panel b: PaqCI cleaves DNA more efficiently when a short hairpin DNA duplex containing the enzyme's target site (but not downstream DNA basepairs corresponding to the adjacent cleavage site) is added to the reaction as a trans-activating factor. In this experiment, lambda phage DNA, with 8 PaqCI sites, is used as a substrate. Samples are quenched at the same time points as previous experiment. PaqCI from 0.125 units to 8 units (2.2 nM to 70 nM enzyme) are used either plus or minus the duplex target site oligonucleotide added in trans. The addition of the duplex target site also increases the performance of the lowest concentration, 0.125 units (2.2 nM enzyme) Panel c: An additional experiment examining cleavage of plasmid substrates validates the results and interpretation above, by indicating (1) that plasmid substrates containing two target sites appear to be cleaved more efficiently than the same plasmid containing only one target site, and (2) that the difference in cleavage efficiency between single- or double-target plasmid substrates is reduced with the same short hairpin containing a target site added in trans. The enzyme's cleavage efficiency against plasmids harboring two target sites does not appear to differ significantly when the target sites (which are separated by approximately 1 kB) are positioned in a head-to-head or head-to-tail arrangement. The enzyme concentration at all time points is 2 units per 50 microliter reaction, corresponding to 17.5 nM enzyme.