Figure 1.
DNA cleavage of supercoiled and linear DNA with one or two R.EcoR124INT recognition sites.
(a) Diagrammatic representation of the DNA substrates used: pUC19 (left) and pTK-neo (right). (b) Restriction assay. The endonuclease was incubated with DNA substrates containing either one or two recognition sites, both supercoiled and linear. Reactions were carried at 37°C and stopped with the addition of 0.5 M EDTA at 1, 5, 15 and 120 minutes. Reactions were run on a 0.8% TBE agarose gel with a 1kb DNA marker (NEB). (c) Densitometry scan of the reaction product of R.EcoR124INT incubated with two-site linear DNA. (d) Kinetics of cleavage of supercoiled DNA with two recognition sites (pUC19). Reaction products were analysed on a 0.8% TBE agarose gel. Reactions were carried at 37°C and stopped with the addition of 0.5 M EDTA at the time points shown over the range 10–300 secs. M represents a 1kb DNA marker (NEB). (e) Quantitation of supercoiled DNA (blue), nicked circle (red) and linear DNA (green) over the time-course of the reaction, by analysis of the data shown in (d).
Figure 2.
(a) Electrophoretic mobility shift assay (EMSA). A 6% native gel was run at 150 V for 2.5 hours. Final concentrations of MTase and DNA were 2 µM. Lanes labelled R1 and R2 correspond to 1∶1 and 2∶1 molar ratios of HsdR to MTase. No difference was observed in the presence of 10 mM MgCl2. (b) Dynamic light scattering. The R1 and R2 complexes had hydrodynamic radii of 6.1 and 6.2 nm, respectively. (c) Sedimentation coefficient distributions of R.EcoR124INT (the R2 complex) plus and minus DNA. Sedimentation velocity data were collected at 285 nm, scanning every 12 minutes at 10°C at 30,000 rpm. Peak sedimentation coefficients for the free enzyme (9.5 S) and the enzyme bound to DNA (10.6 S) were converted to S20,w values of 12.6 S and 14.0 S, respectively.
Figure 3.
Small-angle neutron scattering of R.EcoR124INT.
(a) the SANS profile of R.EcoR124INT measured in H2O (blue triangles) and the SANS of the two HsdR subunits in situ from a sample containing deuterated HsdR subunits and protonated MTase, measured in 40% D2O (red squares). The solid black lines, show the fits from the back-transformed P(r) functions (b) Distance distribution function, P(r), obtained from the scattering profiles shown in (a).
Figure 4.
Multi-phase dummy atom modelling (MONSA) of R.EcoR124INT.
The phase representing the MTase is shown in cyan and the HsdR subunits are in magenta. The model was obtained by simultaneously fitting the SANS data shown in Figure 3. The three views are related by successive 90° rotations around the long axis of the complex.