which are the inherent properties of restriction enzyme? they are present in bacteria as a part of their defence mechanism they are now extensively used for gene cloning they are site specific in their cleavage all of the above.
Answers
The fundamental reaction catalyzed by restriction endonucleases is the hydrolysis of the phosphodiester backbone of DNA. Specifically, the bond between the 3′ oxygen atom and the phosphorus atom is broken. The products of this reaction are DNA strands with a free 3′-hydroxyl group and a 5′-phosphoryl group (Figure 9.33). This reaction proceeds by nucleophilic attack at the phosphorus atom. We will consider two types of mechanism, as suggested by analogy with the proteases. The restriction endonuclease might cleave DNA in mechanism 1 through a covalent intermediate, employing a potent nucleophile (Nu), or in mechanism 2 by direct hydrolysis:
Figure 9.33. Hydrolysis of a Phosphodiester Bond.
Figure 9.33
Hydrolysis of a Phosphodiester Bond. All restriction enzymes catalyze the hydrolysis of DNA phosphodiester bonds, leaving a phosphoryl group attached to the 5′ end. The bond that is cleaved is shown in red.
Mechanism Type 1 (covalent intermediate)
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Mechanism Type 2 (direct hydrolysis)
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Each postulates a different nucleophile to carry out the attack on the phosphorus. In either case, each reaction takes place by an in-line displacement path:
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The incoming nucleophile attacks the phosphorus atom, and a pentacoordinate transition state is formed. This species has a trigonal bipyramidal geometry centered at the phosphorus atom, with the incoming nucleophile at one apex of the two pyramids and the group that is displaced (the leaving group, L) at the other apex. The two mechanisms differ in the number of times the displacement occurs in the course of the reaction.
In the first type of mechanism, a nucleophile in the enzyme (analogous to serine 195 in chymotrypsin) attacks the phosphoryl group to form a covalent intermediate. In a second step, this intermediate is hydrolyzed to produce the final products. Because two displacement reactions take place at the phosphorus atom in the first mechanism, the stereochemical configuration at the phosphorus atom would be inverted and then inverted again, and the overall configuration would be retained. In the second type of mechanism, analogous to that used by the aspartyl and metalloproteases, an activated water molecule attacks the phosphorus atom directly. In this mechanism, a single displacement reaction takes place at the phosphorus atom. Hence, the stereochemical configuration of the tetrahedral phosphorus atom is inverted each time a displacement reaction takes place. Monitoring the stereochemical changes of the phosphorus could be one approach to determining the mechanism of restriction endonuclease action.
A difficulty is that the phosphorus centers in DNA are not chiral, because two of the groups bound to the phosphorus atom are simple oxygen atoms, identical with each other. This difficulty can be circumvented by preparing DNA molecules that contain chiral phosphoryl groups, made by replacing one oxygen atom with sulfur (called a phosphorothioate). Let us consider EcoRV endonuclease. This enzyme cleaves the phosphodiester bond between the T and the A at the center of the recognition sequence 5′-GATATC-3′. The first step in monitoring the activity of the enzyme is to synthesize an appropriate substrate for EcoRV containing phosphorothioates at the sites of cleavage (Figure 9.34). The reaction is then performed in water that has been greatly enriched in 18O to allow the incoming oxygen atom to be marked. The location of the 18O label with respect to the sulfur atom indicates whether the reaction proceeds with inversion or retention of stereochemistry. The analysis revealed that the stereochemical configuration at the phosphorus atom was inverted only once with cleavage. This result is consistent with a direct attack of water at phosphorus and rules out the formation of any covalently bound intermediate