Table 3.

Reactions of Type IIB restriction enzymes on plasmids with one or two recognition sites

Enzyme	Reaction conditionsa	One-site substrateb	Two-site substrateb (site separation)c	Initial rates (nM/min) on:d		Ratio: 2-site rate/1-site rate
				One-site DNA	Two-site DNA
BcgI	40 U/ml in BcgI buffer	pUC19	pDG5 (1810 bp)	0.052 ± 0.004	0.35 ± 0.03	6.7 ± 1.1
AloI	5 U/ml in AloI buffer	pJM1	pJM2 (682 bp)	0.053 ± 0.002	0.20 ± 0.01	3.8 ± 0.3
BaeI	5 U/ml in BaeI buffer	pJM1	pJM2 (703 bp)	0.027 ± 0.004	0.35 ± 0.01	13.0 ± 2.3
BplI	50 U/ml in BplI buffer	pJM1	pJM2 (694 bp)	0.27 ± 0.08	0.30 ± 0.05	1.1 ± 0.6
AlfI	10 U /ml in AlfI buffer	pIS1	pIS2 (1072 bp)	0.31 ± 0.01	1.80 ± 0.15	5.8 ± 0.6
CspCI	10 U/ml in BaeI buffer	pMDS2(a)	pIS1 (1288 bp)	0.021 ± 0.006	0.90 ± 0.03	42.9 ± 14.8
FalI	15 U/ml in BcgI buffer	pJM3	pJM4 (673 bp)	0.12 ± 0.02	0.75 ± 0.08	6.3 ± 1.7.
PpiI	20 U/ml in AloI buffer	pJLV2	pDG5 (842 bp)	0.18 ± 0.01	1.59 ± 0.08	8.8 ± 0.8
BsaXI	5 U/ml in BsaXI buffer (50 mM KOAc)	pDG5	pKW1 (875 bp)	0.13 ± 0.01	0.21 ± 0.02	1.6 ± 0.3
PsrI	10 U/ml in PsrI buffer (66 mM KOAc)	pJM3	pJM4 (681 bp)	0.58 ± 0.03	1.18 ± 0.08	2.0 ± 0.2
BsaXI	5 U/ml in BsaXI buffer with 200 mM KOAc	pDG5	pKW1 (875 bp)	0.025 ± 0.003	0.10 ± 0.01	4.0 ± 0.9
PsrI	10 U/ml in PsrI buffer with 200 mM KOAc	pJM3	pJM4 (681 bp)	0.050 ± 0.005	0.62 ± 0.04	12.4 ± 2.1

^aBuffer compositions are as in Table 2.

^bPlasmid constructs are given in Supplementary Data, Figures S1–S3.

^cSite separations correspond to the shorter of the two centre-to-centre distances in the circular plasmid.

^dThree or more repeats of each reaction were carried out and a mean value calculated for the concentration of supercoiled DNA substrate at each individual time point. The mean values were fitted to a linear slope to give the initial rates shown. Errors on the rates indicate standard errors from the fitting procedure. Compound errors are recorded for the ratios of the rates.