Nucleic Acids Research, 2005, Vol. 33, No. 15 4783 Figure 6. Nucleotide requirement and NTPase activity of PstII. (A) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37 C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, (2)] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. (B) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37 C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. (C) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37 C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, (27)] (D). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37 C and the change in A340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. (E) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above. for CTP over GTP. Cleavage was not observed at any con- centration of UTP (data not shown). Similar sigmoidal cleav- age proﬁles have been observed for EcoP15I with an K1A/T2P of 7 mM (49,51). The ranking order of ATP > CTP > GTP UTP was also observed by measuring the rates of DNA cleav- age at 4 mM rNTP (the appearance of FLL DNA is compared in Figure 6C), although the differences between ATP, CTP and GTP were not as marked as in Figure 6B. Where cleavage