SfiI endonuclease activity is strongly influenced by the non-specific sequence in the middle of its recognition site

Abstract

The SfiI endonuclease cleaves DNA at the sequence GGCCNNNN NGGCC, where N is any base and downward arrow is the point of cleavage. Proteins that recognise discontinuous sequences in DNA can be affected by the unspecified sequence between the specified base pairs of the target site. To examine whether this applies to SFII, a series of DNA duplexes were made with identical sequences apart from discrete variations in the 5 bp spacer. The rates at which SFII cleaved each duplex were measured under steady-state conditions: the steady-state rates were determined by the DNA cleavage step in the reaction pathway. SFII cleaved some of these substrates at faster rates than other substrates. For example, the change in spacer sequence from AACAA to AAACA caused a 70-fold increase in reaction rate. In general, the extrapolated values for k(cat) and K(m) were both higher on substrates with inflexible spacers than those with flexible structures. The dinucleotide at the site of cleavage was largely immaterial. SFII activity is thus highly dependent on conformational variations in the spacer DNA.

Figure 1

Equilibrium binding. Mixtures in SfiI binding buffer contained 10 nM ³²P-labelled duplex, either ATATA (A) or AAAAA (B), 1 nM pAT153 and SfiI endonuclease at one of the following concentrations (increasing from left to right across the gels, as indicated by the wedge): 0, 2.5, 5, 10, 20, 40, 80 and 160 nM. After 30 min at room temperature, the mixtures were subjected to electrophoresis through polyacrylamide. The electrophoretic mobilities of the free DNA and the primary complexes are marked on the left of the gels as F and 1°, respectively.

Figure 2

Cleavage of ATATA. The reactions in SfiI assay buffer at 30°C contained either 0.2 µM ATATA and 0.5 nM SfiI endonuclease (data points marked by inverted triangles) or 1.0 µM ATATA and 2.5 nM SfiI (marked by triangles). The DNA was 5′-end-labelled with ³²P in the top strand. At timed intervals after adding SfiI, samples were withdrawn from the reactions and analysed as in the Materials and Methods to determine the percentage of the substrate that had been cleaved in the top strand. Each data point is the mean from four or more separate experiments. The solid and dashed lines are the optimal fits to linear slopes for the data at 0.2 µM ATATA and at 1.0 µM ATATA, respectively. The optimal fits yielded the reaction velocities given in Table 1.

Figure 3

Cleavage of AAAAA. The reactions in SfiI assay buffer at 30°C contained the concentration of AAAAA (5′-end-labelled with ³²P in the top strand) noted on the x-axis and SfiI endonuclease at a 400-fold lower concentration than the substrate. At timed intervals after adding SfiI, samples were withdrawn from the reactions and analysed as in the Materials and Methods. The increase in the concentration of cleaved DNA with time, during the initial phase of each reaction, was fitted to a linear slope so as to obtain the reaction velocity. The velocities (mol DNA cleaved per min) were then normalised against the enzyme concentrations to give the rates shown on the y-axis. The error bars are the standard deviations from the means from two or more separate experiments.

Figure 4

Single base pair substitutions. The reactions in SfiI assay buffer at 30°C contained 0.5 nM SfiI endonuclease and one of the following duplexes (5′-end-labelled with ³²P in the top strand) at 0.2 µM: AACAA, data points marked by inverted triangles; AAAAC, marked by squares; AAACA, marked by circles. At timed intervals after adding the SfiI, samples were withdrawn from the reactions and analysed as in the Materials and Methods to determine the percent cleavage. The error bars are the standard deviations of the means from three or more separate experiments.

Figure 5

Strand preference. The reactions in SfiI assay buffer at 30°C contained SfiI endonuclease and one of the following oligoduplexes at 0.2 µM: (A) AACAA; (B) AAACA. For the reactions in (A), the concentration of SfiI was 0.5 nM and the AACAA was 5′-end-labelled with ³²P in either the top strand (data points marked by inverted triangles) or the bottom strand (marked by triangles). For the reactions in (B), the concentration of SfiI was 0.1 nM and the AAACA was 5′-end-labelled with ³²P in either the top strand (data points marked by open circles) or the bottom strand (marked by closed circles). At timed intervals after adding SfiI, samples were withdrawn from the reactions and analysed as in the Materials and Methods to determine the percentage of each substrate that had been cleaved in the labelled strand.

Figure 6

Single turnover on AACAA. The reaction in SfiI assay buffer at 30°C contained 10 nM AACAA (5′-end-labelled with ³²P in the top strand) and 20 nM SfiI endonuclease. At timed intervals after adding SfiI, samples were withdrawn from the reactions and analysed as in the Materials and Methods to determine the percent cleavage. The data were fitted to a single exponential and the optimal fit (indicated by the line) was obtained with a value of 1.2 min^–1.