Section 9.3Restriction Enzymes: Performing Highly Specific DNA-Cleavage Reactions

Section 9.3Restriction Enzymes: Performing Highly Specific DNA-Cleavage Reactions

Let us next consider a hydrolytic reaction that results in the cleavage of DNA. Bacteria and archaea have evolved mechanisms to protect themselves from viral infections. Many viruses inject their DNA genomes into cells; once inside, the viral DNA hijacks the cell’s machinery to drive the production of viral proteins and, eventually, of progeny virus. Often, a viral infection results in the death of the host. A major protective strategy for the host is to userestriction endonucleases (restriction enzymes) to degrade the viral DNA on its introduction into a cell. These enzymes recognize particular base sequences, called recognition sequences or recognition sites, in their target DNA and cleave that DNA at defined positions. The most well studied class of restriction enzymes comprises the so-called type II restriction enzymes, which cleave DNA within their recognition sequences. Other types of restriction enzymes cleave DNA at positions somewhat distant from their recognition sites.

Restriction endonucleases must show tremendous specificity at two levels. First, they must cleave only DNAmolecules that contain recognition sites (hereafter referred to as cognate DNA) without cleaving DNA molecules that lack these sites. Suppose that a recognition sequence is six base pairs long. Because there are 46, or 4096, sequences having six base pairs, the concentration of sites that must not be cleaved will be approximately 5000-fold as high as the concentration of sites that should be cleaved. Thus, to keep from damaging host-cell DNA, endonucleases must cleave cognate DNA molecules much more than 5000 times as efficiently as they cleave nonspecific sites. Second, restriction enzymes must not degrade the host DNA. How do these enzymes manage to degrade viral DNA while sparing their own?

The restriction endonuclease EcoRV (from E. coli) cleaves double-stranded viral DNA molecules that contain the sequence 5′-GATATC-3′ but leaves intact host DNA containing hundreds of such sequences. The host DNA is protected by other enzymes called methylases, which methylate adenine bases within host recognition sequences (Figure 9.32). For each restriction endonuclease, the host cell produces a corresponding methylase that marks the host DNA and prevents its degradation. These pairs of enzymes are referred to as restriction-modification systems. We shall return to the mechanism used to achieve the necessary levels of specificity after considering the chemistry of the cleavage process.

Figure 9.32. Protection by Methylation.

Figure 9.32

Protection by Methylation. The recognition sequence for EcoRV endonuclease (left) and the sites of methylation (right) in DNA protected from the catalytic action of the enzyme.

9.3.1. Cleavage Is by In-Line Displacement of 3′ Oxygen from Phosphorus by Magnesium-Activated Water

The fundamental reaction catalyzed by restriction endonucleases is the hydrolysis of the phosphodiester backbone ofDNA. 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:

Image ch9fu7.jpg

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 theT 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 (Figure 9.35).

Figure 9.34. Labeling with Phosphorothioates.

Figure 9.34

Labeling with Phosphorothioates. Phosphorothioates, groups in which one of the nonbridging oxygen atoms is replaced with a sulfur atom, can be used to label specific sites in the DNA backbone to determine the overall stereochemical course of a displacement (more…)

Figure 9.35. Stereochemistry of Cleaved DNA.

Figure 9.35

Stereochemistry of Cleaved DNA. Cleavage of DNA by EcoRV endonuclease results in overall inversion of the stereochemical configuration at the phosphorus atom, as indicated by the stereochemistry of the phosphorus atom bound to one bridging oxygen atom, (more…)

9.3.2. Restriction Enzymes Require Magnesium for Catalytic Activity

Restriction endonucleases as well as many other enzymes that act on phosphate-containing substrates require Mg2+ or some other similar divalent cation for activity. What is the function of this metal?

It has been possible to examine the interactions of the magnesium ion when it is bound to the enzyme. Crystals have been produced of EcoRV endonuclease bound to oligonucleotides that contain the appropriate recognition sequences. These crystals are grown in the absence of magnesium to prevent cleavage; then, when produced, the crystals are soaked in solutions containing the metal. No cleavage takes place, allowing the location of the magnesium ion binding sites to be determined (Figure 9.36). The magnesium ion was found to be bound to six ligands: three are water molecules, two are carboxylates of the enzyme’s aspartate residues, and one is an oxygen atom of the phosphoryl group at the site of cleavage. The magnesium ion holds a water molecule in a position from which the water molecule can attack the phosphoryl group and, in conjunction with the aspartate residues, helps polarize the water molecule toward deprotonation. Because cleavage does not take place within these crystals, the observed structure cannot be the true catalytic conformation. Additional studies have revealed that a second magnesium ion must be present in an adjacent site for EcoRV endonuclease to cleave its substrate.

Figure 9.36. Magnesium Ion Binding Site in ECORV Endonuclease.

Figure 9.36

Magnesium Ion Binding Site in ECORV Endonuclease. The magnesium ion helps to activate a water molecule and positions it so that it can attack the phosphate.

9.3.3. The Complete Catalytic Apparatus Is Assembled Only Within Complexes of Cognate DNA Molecules, Ensuring Specificity

We now return to the question of specificity, the defining feature of restriction enzymes. The recognition sequences for most restriction endonucleases are inverted repeats. This arrangement gives the three-dimensional structure of the recognition site a twofold rotational symmetry (Figure 9.37). The restriction enzymes display a corresponding symmetry to facilitate recognition: they are dimers whose two subunits are related by twofold rotational symmetry. The matching symmetry of the recognition sequence and the enzyme has been confirmed by the determination of the structure of the complex between EcoRV endonuclease and DNA fragments containing its recognition sequence (Figure 9.38). The enzyme surrounds the DNA in a tight embrace. Examination of this structure reveals features that are highly significant in determining specificity.

Figure 9.37. Structure of the Recognition Site of ECORV Endonuclease.

Figure 9.37

Structure of the Recognition Site of ECORV Endonuclease. (A) The sequence of the recognition site, which is symmetric around the axis of rotation designated in green. (B) The inverted repeat within the recognition sequence ofEcoRV (and most other restriction (more…)

Figure 9.38. Structure of the ECORV - Cognate DNA Complex.

Figure 9.38

Structure of the ECORV – Cognate DNA Complex. Image mouse.jpg This view of the structure of EcoRV endonuclease bound to a cognate DNA fragment is down the helical axis of the DNA. The two protein subunits are in yellow and blue, and the DNA backbone is in red. The twofold (more…)

A unique set of interactions occurs between the enzyme and a cognate DNA sequence. Within the 5′-GATATC-3′ sequence, the G and A bases at the 5′ end of each strand and their Watson-Crick partners directly contact the enzyme by hydrogen bonding with residues that are located in two loops, one projecting from the surface of each enzyme subunit (Figure 9.39). The most striking feature of this complex is the distortion of the DNA, which is substantially kinked in the center (Figure 9.40). The central two TA base pairs in the recognition sequence play a key role in producing the kink. They do not make contact with the enzyme but appear to be required because of their ease of distortion. 5′-TA-3′ sequences are known to be among the most easily deformed base pairs. The distortion of the DNA at this site has severe effects on the specificity of enzyme action.

Figure 9.39. Hydrogen Bonding Interactions between ECORV Endonuclease and Its DNA Substrate.

Figure 9.39

Hydrogen Bonding Interactions between ECORV Endonuclease and Its DNA Substrate. Image mouse.jpg One of the DNA-binding loops (in green) of EcoRV endonuclease is shown interacting with the base pairs of its cognate DNA binding site. Key amino acid residues are shown (more…)

Figure 9.40. Distortion of the Recognition Site.

Figure 9.40

Distortion of the Recognition Site. The DNA is represented as a ball-and-stick model. The path of the DNA helical axis, shown in red, is substantially distorted on binding to the enzyme. For the B form of DNA, the axis is straight (not shown).

Specificity is often determined by an enzyme’s binding affinity for substrates. In regard to EcoRV endonuclease, however, binding studies performed in the absence of magnesium have demonstrated that the enzyme binds to all sequences, both cognate and noncognate, with approximately equal affinity. However, the structures of complexes formed with noncognate DNA fragments are strikingly different from those formed with cognate DNA: the noncognate DNA conformation is not substantially distorted (Figure 9.41). This lack of distortion has important consequences with regard to catalysis. No phosphate is positioned sufficiently close to the active-site aspartate residues to complete a magnesium ion binding site (see Figure 9.36). Hence, the nonspecific complexes do not bind the magnesium ion and the complete catalytic apparatus is never assembled. The distortion of the substrate and the subsequent binding of the magnesium ion account for the catalytic specificity of more than 1,000,000-fold that is observed for EcoRV endonuclease despite very little preference at the level of substrate binding.

Figure 9.41. Nonspecific and Cognate DNA within ECORV Endonuclease.

Figure 9.41

Nonspecific and Cognate DNA within ECORV Endonuclease. Image mouse.jpg A comparison of the positions of the nonspecific (orange) and the cognate DNA (red) within EcoRV reveals that, in the nonspecific complex, the DNA backbone is too far from the enzyme to complete (more…)

We can now see the role of binding energy in this strategy for attaining catalytic specificity. In binding to the enzyme, the DNA is distorted in such a way that additional contacts are made between the enzyme and the substrate, increasing the binding energy. However, this increase is canceled by the energetic cost of distorting the DNA from its relaxed conformation (Figure 9.42). Thus, for EcoRV endonuclease, there is little difference in binding affinity for cognate and nonspecific DNA fragments. However, the distortion in the cognate complex dramatically affects catalysis by completing the magnesium ion binding site. This example illustrates how enzymes can utilize available binding energy to deform substrates and poise them for chemical transformation. Interactions that take place within the distorted substrate complex stabilize the transition state leading to DNA hydrolysis.

Figure 9.42. Greater Binding Energy of EcoRV Endonuclease Bound to Cognate Versus Noncognate Dna.

Figure 9.42

Greater Binding Energy of EcoRV Endonuclease Bound to Cognate Versus Noncognate Dna. The additional interactions between EcoRV endonuclease and cognate DNA increase the binding energy, which can be used to drive DNA distortions necessary for forming a (more…)

The distortion in the DNA explains how methylation blocks catalysis and protects host-cell DNA. When a methyl group is added to the amino group of the adenine nucleotide at the 5′ end of the recognition sequence, the methyl group’s presence precludes the formation of a hydrogen bond between the amino group and the side-chain carbonyl group of asparagine 185 (Figure 9.43). This asparagine residue is closely linked to the other amino acids that form specific contacts with the DNA. The absence of the hydrogen bond disrupts other interactions between the enzyme and the DNA substrate, and the distortion necessary for cleavage will not take place.

Figure 9.43. Methylation of Adenine.

Figure 9.43

Methylation of Adenine. The methylation of adenine blocks the formation of hydrogen bonds between EcoRV endonuclease and cognate DNA molecules and prevents their hydrolysis.

9.3.4. Type II Restriction Enzymes Have a Catalytic Core in Common and Are Probably Related by Horizontal Gene Transfer

Image tree.jpg Type II restriction enzymes are prevalent in Archaea and Eubacteria. What can we tell of the evolutionary history of these enzymes? Comparison of the amino acid sequences of a variety of type II restriction endonucleases did not reveal significant sequence similarity between most pairs of enzymes. However, a careful examination of three-dimensional structures, taking into account the location of the active sites, revealed the presence of a core structure conserved in the different enzymes. This structure includes β strands that contain the aspartate (or, in some cases, glutamate) residues forming the magnesium ion binding sites (Figure 9.44).

Figure 9.44. A Conserved Structural Core in Type II Restriction Enzymes.

Figure 9.44

A Conserved Structural Core in Type II Restriction Enzymes. Image mouse.jpg Four conserved structural elements, including the active-site region (in blue), are highlighted in color in these models of a single monomer from each dimeric enzyme. The positions of the amino (more…)

These observations indicate that many type II restriction enzymes are indeed evolutionary related. Analyses of the sequences in greater detail suggest that bacteria may have obtained genes encoding these enzymes from other species by horizontal gene transfer, the passing between species of pieces of DNA (such as plasmids) that provide a selective advantage in a particular environment. For example, EcoRI (from E. coli) and RsrI (from Rhodobacter sphaeroides) are 50% identical in sequence over 266 amino acids, clearly indicative of a close evolutionary relationship. However, these species of bacteria are not closely related, as is known from sequence comparisons of other genes and other evidence. Thus, it appears that these species obtained the gene for this restriction endonuclease from a common source more recently than the time of their evolutionary divergence. Moreover, the gene encoding EcoRI endonuclease uses particular codons to specify given amino acids that are strikingly different from the codons used by most E. coli genes, which suggests that the gene did not originate in E. coli. Horizontal gene transfer may be a relatively common event. For example, genes that inactivate antibiotics are often transferred, leading to the transmission of antibiotic resistance from one species to another. For restriction-modification systems, protection against viral infections may have favored horizontal gene transfer.

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Copyright © 2002, W. H. Freeman and Company.

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