There is little amino acid sequence homology between the nuclease and methylase within a restriction/modification system, even among the regions responsible for recognition. Among restriction enzymes, exact isoschizomers isolated from bacteria of the same genus can show little or no similarity in their methylation sensitivity, digestion optima or primary sequence except for a limited PD…D/EXK motif involved in catalysis. However, this common motif has been found in Type II, IIb, IIe, IIs and in intron-encoded restriction enzymes (1,2).

Despite the lack of primary sequence homology, three-dimensional structure among Type II homodimers is similar for those enzymes where crystallography data is available. In general, the holoenzyme dimer resembles a "U" shape, with each side constituting a monomer containing both recognition and catalytic domains with an overlapping bridging domain at the bottom. The DNA is bound between the two subunits. Fok I, the most studied Type IIs enzyme, appears to exist primarily as a monomer but transiently forms a similar dimer at the recognition site (3).

Restriction endonucleases bind dsDNA both specifically and non-specifically. After binding at a non-cognate sequence, several enzymes have been shown to locate their targets through linear diffusion. For example, EcoR I diffuses along linear DNA at a rate of approximately 7 x 10⁶bp s^-1 (4) and EcoR V diffuses at approximately 1.7 x 10⁶bp s^-1 (5). During this process a large number of water molecules appear to fill the spaces between the enzyme and the DNA. Once the cognate (recognition) sequence is found, much of the water is excluded as a highly redundant number of contacts evolve between the enzyme and the bases and phosphodiester backbone of the DNA. In the case of EcoR I, 50 water molecules are excluded at the cognate site (6). Generally, 2-3 non-specific bases on either side of the target sequence are required for proper recognition. Conformational changes occur in both the enzyme and DNA as the specific complex forms. The resulting induced fit positions the catalytic center in reactive proximity to the substrate. For most enzymes studied to date, this is able to occur in the absence of Mg²⁺.

Using the known co-crystal structures of enzymes bound to their cognate sequences and substitution experiments in the enzyme or DNA for a limited number of additional enzymes, a mechanism for DNA cleavage has been postulated. Evidence for most enzymes studied to date supports a substrate assisted catalysis model (7). In this model, conserved amino acids at the catalytic site bind Mg²⁺ and position it near the scissile phosphate. Hydrolysis begins by in-line nucleophilic attack of an activated water molecule. The phosphate 3´ of the scissile phosphorous has been shown to play some role in catalysis, most likely in activating the water, as greatly reduced cleavage occurs when a methylphosphonate (8) or phosphothioate (9) occupy this position. A conserved lysine and/or a Mg²⁺ also may be involved in activating the water and stabilizing the pentavalent transition state produced at the scissile phosphorous (10). Inversion occurs as the 3´-OH leaving group is protonated by a Mg²⁺-bound water upon exit (Figure 1.3).

Regardless of the mechanism of action, all restriction enzymes share two common features, a requirement for Mg²⁺, and 5´-phosphate and 3´-OH products. Some enzymes may also need AdoMet or ATP, and/or binding of a second recognition sequence to an allosteric site on the enzyme as a requirement for, or a stimulator of, cleavage.

Note: The following image requires Macromedia Shockwave to view. If you do not see the animated image, you may download a copy of Shockwave at: http://www.macromedia.com/shockwave/.

Figure 1.3. A possible general mechanism for the hydrolysis of phosphodiester bonds in DNA by restriction enzymes. Shown is the scissile phosphorous and cleavage of a single strand. The resulting 5´-phosphorous and DNA fragment are represented in red. The resulting 3´-hydroxyl fragment is in blue. Oxygen and hydrogen is represented in black and light blue respectively. After clicking on "Go", the mechanism will automatically repeat, clicking on "Pause" will stop the cartoon. Each click of "Step" will advance the cartoon one frame.

References

1. Wilson, G.G. and Murray, N.E. (1991) Restriction and modification systems. Annu. Rev. Genet. 25, 585.
2. Stahl, F. et al. (1998) The mechanism of DNA cleavage by the type II restriction enzyme EcoR V: Asp36 is not directly involved in DNA cleavage but serves to couple indirect readout to catalysis. Biol. Chem. 379, 467.
3. Bitinaite, J. et al. (1998) Fok I dimerization is required for DNA cleavage. Proc. Natl. Acad. Sci.USA 95, 10570.
4. Ehbrecht, H.J. et al. (1985) Linear diffusion of restriction endonucleases on DNA. J. Biol. Chem. 260, 6160.
5. Jeltsch, A. and Pingoud, A. (1998) Kinetic characterization of linear diffusion of the restriction endonuclease EcoR V on DNA. Biochem. 37, 2160
6. Robinson, C.R. and Sligar, S.G. (1998) Changes in solvation during DNA binding and cleavage are critical to altered specificity of the EcoR I endonuclease. Proc. Natl. Acad. Sci USA 95, 2186.
7. Pingoud, A. and Jeltsch, A. (1997) Recognition and cleavage of DNA by type-II restriction endonucleases. Eur. J. Biochem. 246, 1.
8. Jeltsch, A. et al. (1995) Evidence for substrate-assisted catalysis in the DNA cleavage of several restriction endonucleases. Gene 157, 157.
9. Jeltsch, A. et al. (1993) Substrate-assisted catalysis in the cleavage of DNA by the EcoR I and EcoR V restriction enzymes Proc. Natl. Acad. Sci. USA 90, 8499.
10. Sam, M.D. and Perona, J.J. (1999) Catalytic roles of divalent, metal ions in phosphoryl transfer by EcoR V endonuclease. Biochem. 38, 6576-6586.

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