Posts Tagged ‘Restriction enzyme’

Targeted Nucleases

Curator: Larry H Bernstein, MD, FCAP

A REVIEW of 3 published works

Targeted nucleases: spreading the joy
Nature Methods 10, 179 (2013)

Published online 27 February 2013
New RNA-guided endonucleases (RGENs) are directed to their target sites
  • by a complementary RNA molecule.
In contrast to previous tools,
  • zinc-finger nucleases (ZFNs) and
  • transcription activator–like effector nucleases (TALENs),
the RGEN nuclease component itself does not require re-engineering to
  • target a new sequence.
The ability to manipulate DNA has led to a new genetics.
Professor of Genetics at Washington University’s School of Medicine
Backgrounders – introduction to issues of current interest

Restriction Endonucleases

In 1980, geneticists used the relatively new technique of gene splicing, which we will describe in this chapter, to introduce
  • the human gene that encodes interferon into a bacterial cell’s genome.
Interferon is a rare blood protein that increases human resistance to viral infection, and medical scientists have been interested in its possible usefulness in cancer therapy. Purification of the large amounts of interferon required for clinical testing would have been prohibitively expensive at the time.   Introducing the gene responsible for its production into a bacterial cell made that possible. The cell that had
  • acquired the human interferon gene proceeded to produce interferon at a rapid rate, and to grow and divide.

The  millions of interferon-producing bacteria growing in the culture were all descendants of the cell that had originally received the human interferon gene.

The Advent of Genetic Engineering
The human insulin gene has also been cloned in bacteria, and now insulin can be manufactured at little expense. Furthermore, cloning and related molecular techniques are needed to provide basic information about how genes are put together and regulated.
The essence of genetic engineering is
  • the ability to cut DNA into recognizable pieces and rearrange those pieces in different ways.
In the interferon experiment,
  • a piece of DNA carrying the interferon gene was
  • inserted into a plasmid,which
    • carried the gene into a bacterial cell.
Most other genetic engineering approaches bring the gene of interest into the target cell by first incorporating it into a plasmid or an infective virus.
This cutting is performed
  • by enzymes that recognize and
  • cleave specific sequences of nucleotides in DNA.
Discovery of Restriction Endonucleases
Scientific discoveries often have their origins in seemingly unimportant observations that receive little attention by researchers before their general significance is appreciated. In the case of genetic engineering, the original observation was that bacteria use enzymes to defend themselves against viruses.
Most organisms eventually evolve means of defending themselves from predators and parasites, and bacteria are no exception. Among the natural enemies of bacteria are bacteriophages, viruses that infect bacteria and multiply within them. At some point, they cause the bacterial cells to burst, releasing thousands more viruses.
Some types of bacteria have acquired powerful weapons against these viruses: they contain enzymes called restriction endonucleases
  • that fragment the viral DNA as soon as it enters the bacterial cell.
Many restriction endonucleases recognize
  1. specific nucleotide sequences in a DNA strand,
  2. bind to the DNA at those sequences, and
  3. cleave the DNA at a particular place within the recognition sequence.
Why don’t restriction endonucleases cleave the bacterial cells’ own DNA as well as that of the viruses?
  • bacteria modify their own DNA, using other enzymes known as methylases to add methyl (CH3) groups
  • to some of the nucleotides in the bacterial DNA.
When nucleotides within a restriction endonuclease’s recognition sequence have been methylated,
  • the endonuclease cannot bind to that sequence.
  • the bacterial DNA is protected from being degraded at that site.
  • but viral DNA has not been methylated, and therefore
    • is not protected from enzymatic cleavage.
How Restriction Endonucleases Cut DNA
The sequences recognized by restriction endonucleases are
  • typically four to six nucleotides long, and
  • they are often palindromes.
    • the nucleotides at one end of the recognition sequence are complementary to those at the other end, so that
    • the two strands of the DNA duplex have the same nucleotide sequence running in opposite directions for the length of the recognition sequence.

Two important consequences arise from this arrangement of nucleotides to be discussed.

Biochemistry. 5th edition.
Berg JM, Tymoczko JL, Stryer L.
Section 9.3  Restriction Enzymes: Performing Highly Specific DNA-Cleavage Reactions
Bacteria and archaea have evolved mechanisms to protect themselves from viral infections so that viruses inject their DNA genomes into cells and the viral DNA hijacks the cell’s machinery A major protective strategy for the host is to use restriction endonucleases (restriction enzymes) to degrade the viral DNA. These  particular base sequences the enzymes recognize are called recognition sequences or recognition sites.
  • theycleave that DNA at defined positions.
  • The most well studied class are the so-called type II restriction enzymes.
Restriction endonucleases must show tremendous specificity at two levels.
  • First, they must cleave only DNA molecules that contain recognition sites (hereafter referred to as cognate DNA) without cleaving DNA molecules that lack these sites.
    •  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.
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)
Mechanism Type 2 (direct hydrolysis)
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:
  • 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.
The incoming nucleophile attacks the phosphorus atom, and
  • a pentacoordinate transition state is formed.
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).
Stereochemistry of Cleaved DNA.
Cleavage of DNA by EcoRV endonuclease results in overall inversion of the stereochemical configuration at the phosphorus atom.
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:
  1. three are water molecules,
  2. two are carboxylates of the enzyme’s aspartate residues, and
  3. 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.
Cleavage does not take place within these crystals. But a second magnesium ion must be present in an adjacent site for EcoRV endonuclease to cleave its substrate.
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
Specificityis 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.

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 of EcoRV
 Structure of the ECORV – Cognate DNA Complex.
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.
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.
Hydrogen Bonding Interactions between ECORV Endonuclease and Its DNA Substrate.
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.
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.
  • 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
Nonspecific and Cognate DNA within ECORV Endonuclease.
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
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.
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.
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.
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
 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).
 A Conserved Structural Core in Type II Restriction Enzymes.
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.
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.
  • 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.
  • 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.
    • 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.
Biochemistry. 5th edition.
Berg JM, Tymoczko JL, Stryer L.
New York: W H Freeman; 2002.
  • Cleavage Is by In-Line Displacement of 3′ Oxygen from Phosphorus by Magnesium-Activated Water
  • Restriction Enzymes Require Magnesium for Catalytic Activity
  • The Complete Catalytic Apparatus Is Assembled Only Within Complexes of Cognate DNA Molecules, Ensuring Specificity
  • Type II Restriction Enzymes Have a Catalytic Core in Common and Are Probably Related by Horizontal Gene Transfer
By Richard Wheeler (Zephyris) 2007. Image of E...

By Richard Wheeler (Zephyris) 2007. Image of EcoRV homodimer in complex with a DNA substrate. From . (Photo credit: Wikipedia)

HindIII restriction endonuclease in complex wi...

HindIII restriction endonuclease in complex with cognate DNA (Photo credit: Wikipedia)

English: 3d surface model of HindIII dimer com...

English: 3d surface model of HindIII dimer complexed with a DNA fragment from PDB 2E52. Ref.: Watanabe, N., Sato, C., Takasaki, Y., Tanaka, I. Crystal structural analysis of HindIII restriction endonuclease in complex with cognate DNA at 2.0 angstrom resolution to be published (Photo credit: Wikipedia)

English: BglII active site containing residues...

English: BglII active site containing residues that coordinate to a metal ion and water molecules including the nucleophilic water that breaks the scissile phosphodiester bond at the recognition site. (Photo credit: Wikipedia)

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