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What is the distinction between deoxyribonucleases and restriction enzymes?


Both deoxyribonucleases (DNases) and restriction enzymes are endonucleases (some DNases can be exonucleases).

They both break the bonds between nucleotides. Therefore, what is the difference between the two?

My original thought was that restriction enzymes, unlike DNases, cut at specific sequences of DNA. Then this raises the question of whether restriction enzymes are just a category of DNases, but I have found no sources to back that up. Also, according to this Wikipedia article:

There are more than 900 restriction enzymes, some sequence specific and some not…

This further erodes the distinction I had created between the two types of nucleases.


This paragraph from Lehninger's Principles of Biochemistry (in my opinion the best textbook on biochemistry) is very didactic:

DNA Is Degraded by Nucleases

To explain the enzymology of DNA replication, we first introduce the enzymes that degrade DNA rather than synthesize it. These enzymes are known as nucleases, or DNases if they are specific for DNA rather than RNA. Every cell contains several different nucleases, belonging to two broad classes: exonucleases and endonucleases. Exonucleases degrade nucleic acids from one end of the molecule. Many operate in only the 5'→3' or the 3'→5' direction, removing nucleotides only from the 5' or the 3' end, respectively, of one strand of a double stranded nucleic acid or of a single-stranded DNA. Endonucleases can begin to degrade at specific internal sites in a nucleic acid strand or molecule, reducing it to smaller and smaller fragments. A few exonucleases and endonucleases degrade only single-stranded DNA. There are a few important classes of endonucleases that cleave only at specific nucleotide sequences (such as the restriction endonucleases that are so important in biotechnology; see Chapter 9, Fig. 9-3).

We can conclude that restriction endonucleases (or restriction enzymes) are a type of endonuclease, and that endonucleases are a type of DNAse. So, all restriction enzymes are DNAses, but not all DNAses are restriction enzymes.

Besides that, we can conclude that your initial statement ("Both deoxyribonucleases (DNases) and restriction enzymes are endonucleases") is wrong.

For a more complete table have a look at Molecular Biology of Nucleases, page 18.

Sources:

  • Lehninger, A., Nelson, D. and Cox, M. (2000). Principles of biochemistry. New York: Worth Publishers.
  • Mishra, N. (1995). Molecular biology of nucleases. Boca Raton [u.a.]: CRC Press.

Nucleic Acid Structure and Properties of DNA

23.6 Degradation of DNA

Nucleases

A variety of enzymes break phosphodiester bonds in nucleic acids deoxyribonucleases (DNases) cleave DNA and ribonucleases (RNases) cleave RNA. DNases usually are specific for single- or double-stranded DNA although some DNases can cleave both. DNases can act as exonucleases in which they remove one nucleotide at a time from either the 3' or 5' end of the strand. Other DNases function as endonucleases and are specific for cleaving between particular pairs of bases.

Restriction Enzymes

An important class of DNA endonucleases are restriction enzymes (restriction endonucleases) that recognize specific sequences of bases in DNA (a restriction site) and make two cuts, one in each strand that generates fragments of double-stranded DNA. Microorganisms use their restriction enzymes to degrade any foreign DNA that may enter the cell in the form of viruses, plasmids, or naked DNA. Hundreds of different restriction enzymes have been isolated from microorganisms these enzymes generally recognize sequences of four, six, eight, or rarely more bases that have an axis of symmetry ( Table 23-1 ). Restriction sites that have an axis of symmetry are called palindromes, which means that the restriction site can be rotated 180 degrees and the sequence of bases will remain the same.

TABLE 23-1 . Some Restriction Endonucleases and Their Cleavage Sites *

Restriction enzymes can cut DNA in either of two ways. Each strand can be cleaved along the axis of symmetry generating fragments of double-stranded DNA that have blunt (flush) ends. Symmetrical cuts that are staggered around the axis of symmetry generate fragments of double-stranded DNA that have single-stranded cohesive ends ( Figure 23-11 ). Fragments of DNA with cohesive ends are useful for constructing novel DNA molecules, which is the goal of recombinant DNA (rDNA) technology.

FIGURE 23-11 . Two types of cuts made by restriction enzymes. The arrows indicate the cleavage sites. The dashed line is the axis of symmetry of the sequence.

Since a restriction enzyme recognizes a unique sequence, the number of cuts and the number of DNA fragments depends on the size of the molecule. In general, restriction sites consisting of four bases will occur more frequently by chance than sites consisting of six or eight bases. Thus, four-cutters will generate more fragments than six-cutters which, in turn, will generate more fragments than eight-cutters.

A particular restriction enzyme generates a unique family of fragments for a particular DNA molecule.

Figure 23-12 a shows the positions of restriction sites in bacteriophage lambda (λ) DNA for the restriction enzymes EcoRI and BamHI. This arrangement of restriction sites is known as a restriction map. The family of fragments generated by one or more restriction enzymes is detected by agarose gel electrophoresis that separates DNA molecules according to their molecular weights ( Figure 23-12b ).

FIGURE 23-12 . (a) Restriction maps of λ DNA for EcoRI and BamHI nucleases. The vertical bars indicate the sites of cutting. The top numbers indicate the percentage of the total length of DNA measured from the gene A end of the molecule. The bottom numbers are the lengths of each fragment, again expressed as percentage of total length. (b) Gel electrophoretogram of EcoRI and BamHI restriction enzyme digests of λ DNA. The bands labeled “cohered ends” contain molecules consisting of two terminal fragments, joined by the normal cohesive ends of λ DNA. Numbers indicate fragments in order from largest 1 to smallest 6. Bands 5 and 6 of the BamHI digest are not resolved.


Restriction Enzymes: Types & Examples

One of the most important steps in molecular biology, especially molecular genetics and analysis, is the isolation of DNA from the human genome and make many copies of it. Now, these copies can be utilized for further analysis of whatsoever type.

A key event in the development of molecular genetics methodology has been the discovery of Restriction Enzymes, also known as Restriction Endonucleases.

Introduction

A restriction enzyme is a kind of nuclease enzyme which is capable of cleaving double-stranded DNA. The enzymes may cleave DNA at random or specific sequences which are referred to as restriction sites. The recognition sites are palindromic in origin, that is, they are the sequences which are read the same forward and backward.

These restriction enzymes are produced naturally by bacteria. The bacterial species use it as a form of defense mechanism against viruses. However, in bacteria, restriction enzymes are present as a part of a combined system called the restriction modification system. The bacterial species modify their own DNA with the help of enzymes which methylate it. This particular process of methylation of bacterial DNA protects it from cleavage from its own restriction endonucleases.

Types

There are two different kinds of restriction enzymes:

  1. Exonucleases: restriction exonucleases are primarily responsible for hydrolysis of the terminal nucleotides from the end of DNA or RNA molecule either from 5’ to 3’ direction or 3’ to 5’ direction for example- exonuclease I, exonuclease II, etc.
  2. Endonuclease: restriction endonucleases recognize particular base sequences (restriction sites) within DNA or RNA molecule and catalyze the cleavage of internal phosphodiester bond for exEcoRI, Hind III, BamHI, etc.

History

The first restriction enzyme to be discovered was Hind II in the year 1970. In 1978, Daniel Nathans, Werner Arber, and Hamilton O. Smith were awarded the Nobel Prize for Physiology or Medicine.

Restriction Enzyme Nomenclature

The very name of the restriction enzymes consists of three parts:

  1. An abbreviation of the genus and the species of the organism to 3 letters, for example- Eco for Escherichia coli identified by the first letter, E, of the genus and the first two letters, co, of the species.
  2. It is followed by a letter, number or combination of both of them to signify the strain of the species.
  3. A Roman numeral to indicate the order in which the different restriction-modification systems were found in the same organism or strain per se.

Classification of Restriction Endonucleases

Based on the types of sequences identified, the nature of cuts made in the DNA, and the enzyme structure, there are three classes:

  1. Type I restriction enzymes,
  2. Type II restriction enzymes, and
  3. Type III restriction enzymes.

A. Type I Restriction Enzymes

  • Type I restriction enzymes possess both restriction and modification activities. In this case, the restriction will depend upon the methylation status of the target DNA sequence.
  • Cleavage takes place nearly 1000 base pairs away from the restriction site.
  • The structure of the recognition site is asymmetrical. It is composed of 2 parts. One part of the recognition site is composed of 3-4 nucleotides while the other one contains 4-5 nucleotides. The two parts are separated by a non-specific spacer of about 6-8 nucleotides.
  • For their function, the type I restriction enzymes require S- adenosylmethionine (SAM), ATP, and Mg 2+
  • They are composed of 3 subunits, a specificity subunit which determines the recognition site, a restriction subunit, and a modification subunit.

B. Type II Restriction Enzymes

  • Two separate enzymes mediate restriction and modification. Henceforth, DNA can be cleaved in the absence of modifying enzymes. Although the target sequence identified by the two enzymes is the same, they can be separately purified from each other.
  • The nucleotides are cleaved at the restriction site only. The recognition sequence is rotationally symmetrical, called palindromic sequence. The specific palindromic site can either be continuous (e.g., KpnI identifies the sequence 5´-GGTACC-3´) or non-continuous (e.g., BstEII recognizes the sequence 5´-GGTNACC-3´, where N can be any nucleotide)
  • These require Mg 2+ as a cofactor but not ATP.
  • They are required in genetic mapping and reconstruction of the DNA in vitro only because they identify particular sites and cleave at those sites only.

How they work:

  • The type II restriction enzymes first establish non-specific contact with DNA and bind to them in the form of dimmers.
  • The target sequence is then detected by a combination of two processes. Either the enzyme diffuses linearly/ slides along the DNA sequence over short distances or hops/ jumps over long distances.
  • Once the target sequence is located, various conformational changes occur in the enzyme as well as the DNA. These conformational changes, in turn, activate catalytic center.
  • The phosphodiester bond is hydrolyzed, and the product is released.

Structures of free, nonspecific, and specific DNA-bound forms of BamHI

C. Type III Restriction Enzyme

  • The type III enzymes recognize and methylate the same DNA sequence. However, they cleave nearly 24-26 base pairs away.
  • They are composed of two different subunits. The recognition and modification of DNA are carried out by the first subunit- ‘M’ and the nuclease activity is rendered by the other subunit ‘R’.
  • DNA cleavage is aided by ATP as well as Mg 2+ whereas SAM is responsible for stimulating cleavage.
  • Only one of the DNA strand is cleaved. However, to break the double-stranded DNA, two recognition sites in opposite directions are required.

Star Activity

Some restriction enzymes are capable of cleaving recognition sites which are similar to but not identical to the defined recognition sequence under non-standard reaction conditions (low ionic strength, high pH).

Isoschizomers, Neoschizomers, and Isocaudomers

  • Isoschizomers are the restriction enzymes which recognize and cleave at the same recognition site. For example, SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers of each other.
  • Neoschizomers are the restriction enzymes which recognize the same site and have a different cleavage pattern. For example, SmaI (GGG/CCC) and XmaI (G/GGCCC) are neoschizomers of each other.
  • Isocaudomers are the restriction enzymes which recognize slightly different sequences but produce the same ends. For example, both Sau3a and BamHI render a 5’-GATC-3’ sticky end although both have different recognition sequences.

Cleavage Patterns

Cleavage patterns of HindIII, SmaI, EcoRI, and BamHI are described as below. Most of the enzymes recognize sequences which are 4 to 6 base pairs long. However, they can also be up to 8 base pairs in length.

The process of cleavage of DNA by the restriction enzyme culminates with the formation of either sticky ends or blunt ends.

The blunt-ended fragments can be joined with the DNA fragment only with the aid of linkers and adapters.


What are Restriction Enzymes?

A restriction enzyme, more commonly referred to as a restriction endonuclease, has the ability to cleave DNA molecules into small fragments. The cleaving process occurs near or at a special recognition site of the DNA molecule called a restriction site. A recognition site is typically composed of 4-8 base pairs. Depending on the site of cleavage, restriction enzymes can be of four (04) different types: Type I, Type II, Type III and Type IV. Other than the site of cleavage, factors such as composition, requirement of co-factors and the condition of the target sequence are taken into consideration when differentiating restriction enzymes into four groups.

During the cleavage of DNA molecules, the cleaving site can be either at the restriction site itself or at a distance from the restriction site. Restriction enzymes create two incisions through each of the sugar-phosphate backbone in the double helix of DNA.

Figure 02: Restriction Enzymes

Restriction enzymes are mainly found in Achaea and bacteria. They utilize these enzymes as a defense mechanism against the invading viruses. The restriction enzymes cleave the foreign (pathogenic) DNA but not their own DNA. Their own DNA is protected by an enzyme known as methyltransferase, which makes modifications in the host DNA and prevents cleavage.

Type I restriction enzyme possesses a cleaving site which is away from the recognition site. Functioning of the enzyme requires ATP and the protein, S-adenosyl-L-methionine. Type I restriction enzyme is considered to be multifunctional due to the presence of both restriction and methylase activities. Type II restriction enzymes cleave within the recognition site itself or at a closer distance to it. It only requires magnesium (Mg) for its function. Type II restriction enzymes have only one function and are independent of methylase.


What’s the difference between a restriction enzyme and CRISPR?

CRISPR Cas9 is a restriction enzyme. There are many types of restriction enzymes, and each one cuts the DNA at a specific place. CRISPR is the name of a family of DNA sequences found in the DNA of many prokaryotes which CRISPR Cas9 can cleave, making it a useful marker for a lot of things.

Think of DNA as a chain made of different padlocks. Restriction enzymes are the keys. Each key can open one lock. CRISPR is a brand of lock, a brand that has a master key for all of their locks called CRISPR Cas9. Anywhere along this chain that you find a CRISPR lock, you can use the Cas9 protein to open it.

This is the only correct answer. Cas9 is a type V restriction enzyme.

thank you so much this helped a lot I was trying to find the difference on google and it brought up your comment thank you

Uncontrollable snips that look for a set sequence anywhere vs a controlled break at a specific sequence point using a guide.

Restriction enzyme looks for a certain sequence, whenever it finds it no matter where it will snip around it, nothing added nor removed. Because its not controlled you might get things snipped you don't want snipped, buffers and temperature can help here.

CRISPR instead relies on an RNA sequence (guide RNA) complimentary to the target, when the guide binds CRISPR makes a double strand break, bringing in sequence errors like insertions and deletions, however this can be accounted for with a replacement strand.

ahhh okay so this is what makes CRISPR better for genetic modification right? It’s more precise

Generally when we're talking about 'restriction enzymes' we're referring to the historical ones that recognize a single sequence, generally 6 or 8 nucleotides. They are hard-wired to cut only a single sequence (though that sequence may appear in many places). If you hear about "EcoR1" or genetic engineering, you're likely hearing about these 'Type II' restriction enzymes.

The magic of Cas9 is that it is ⟊ssette based'--if you provide a companion RNA (that is made according to a few rules, but BASICALLY can target any sequence at all) then whatever DNA sequence matches that RNA will receive a cut. Further, the 'matching target' for Cas9 is relatively large, so much so that you can target a SINGLE gene in the 1 billion nucleotides of human DNA. It's not always perfect, but that's another story.

FORMALLY, humans have chosen to classify it as a particular kind of the huge and diverse family of DNA cutting machines, hence saying it 'is' a restriction enzyme is also correct but it's a very distinct type with awesome properties and potential.

thank you for clarifying lol I’ve learnt abit about it at school but they didn’t make it clear that cas9 was also a restriction enzyme. They sort of teach it as a whole separate thing lol but yeah this makes sense :))

Crispr stands for clustered regularly interspaced short palindromic repeats. They are dna sequences found in prokaryotes (bacteria) that contain the same sequences found in viruses that attack those bacteria. Bacteria will detect these palindromic sequences in an invader virus's dna and add them to its own dna in these crispr sequences. The bacteria then uses them as a kind of index, and the Cas-9 protein (crispr associated protein #9) uses these sequences to find and cut up viral dna that matches those found in it's crispr sequences. It's fascinating, a kind of adaptive immune defence for an individual bacteria. If you look into how multicellular life fights and adapts to infections there are parallels. Some might consider this a kind of convergent evolution.

A restriction enzyme is any enzyme that cuts dna it finds. They utilize palindromic sequences to identify places where they can work. Cas-9 is a type of restriction enzymes, but there are many more. Cas-9 is very powerful, however, as it is able to be used for any given palindromic sequence.


What Is DNA Ligase?

The sticky ends of the fragments produced by restriction enzymes are useful in a laboratory setting. They can be used to join DNA fragments from both different sources and different organisms. The fragments are held together by hydrogen bonds. From a chemical perspective, hydrogen bonds are weak attractions and are not permanent. Using another type of enzyme however, the bonds can be made permanent.

DNA ligase is a very important enzyme that functions in both the replication and repair of a cell's DNA. It functions by helping the joining of DNA strands together. It works by catalyzing a phosphodiester bond. This bond is a covalent bond, much stronger than the aforementioned hydrogen bond and able to hold the different fragments together. When different sources are used, the resulting recombinant DNA that is produced has a new combination of genes.


What is the difference between single digestion and double digestion when discussing restriction enzymes?

NOTE: in this answer, for RE's (Restriction Enzymes) read Endonucleases.

NOTE2: I assume you are familiar with DNA fingerprinting. If not, see here and here.

RE's are highly specific for the DNA-sequence they splice: it is almost invariably a predetermined Palindromic sequence.
For instance, Hin DIII will only make a cut in the sequence:

5' #-># A #color(red)/# AGCTT #-># 3'
3' #larr# TTCGA #color(red)/# A #larr# 5'

The amount of bases in this palindorme is 6.
I'm not going into statistical analysis, but it is safe to assume this will result in less cuts than for instance the activity of Sau3A:

5' #-># ---- #color(red)/# GATC---- #-># 3'
3' #larr# ----CTAG #color(red)/# ---- #larr# 5'

If you have an amount of (total nuclear) DNA, say, from a human for "fingerprinting", digesting (splicing, or cutting) it with Sau3A might not lead to the desired result: you will get too many small fragments resulting in an unintelligible "smear" across your Electrophoresis Gel slab.

Cutting with Hin DIII will result in a few large bands that can be tell-tale, but you would like to have more certainty.

So, cutting with TWO different restriction enzymes, like a mixture of Hin DIII (5' #-># A #color(red)/# AGCTT #-># 3') and EcoR1 (5' #-># G #color(red)/# AATTC #-># 3') might do the trick.

You can run into trouble with this however: DNA is fairly inert and robust, but RE's are not. Since they are extracted from different organisms (bacteria), each RE will have its own optimum environment within the Host Cell. Therefore, each RE will have it's own optimum buffer-mixture (think of pH if nothing else) , and therein lies the problem.

Although in the laboratory it has been found that most RE's are fairly happy with TRIS-based buffers (Tris-HydroxyMethyl AminoMethane), such as TBE (Tris/Borate/EDTA), each RE will need tweaking of the chosen buffer for optimum activity. Other factors, such as temperature, can also have an influence.

So, if the two RE's are compatible bufferwise you might do a double digest in one go, but depending on the compatibility of your two RE's of choice, you might need to do it in two phases: stop the reaction of the first digest (heat-shock), purify the DNA-digest, then do the second digest.

But as by the time of writing over 3000 RE's have been identified, and more than 600 are commercially available, it is not for me to give you an exhaustive list of combinations.

In the 80's we got most of our RE's from either Bethesda labs (USA), New England Biolabs (USA) or Boehringer Ingelheim (Germany), and they might give you a list of compatible RE's and their buffers for double digestion.


What are Type II Restriction Enzymes?

Type II restriction enzymes contain two identical subunits within their structure. Homodimers are formed by type II restriction enzymes with the recognition sites. The recognition sites are typically palindromic and are undivided. It has a length of 4-8 base pairs. Unlike type I, Type II restriction enzyme’s cleavage site is present at the recognition site or present at close distance to the recognition site.

Figure 02: Type II Restriction Enzymes

These restriction enzymes are biochemically significant and are widely available commercially. For its activation, it requires only Mg 2+ . It doesn’t have a methylation activity and only provides the function of restriction activity. These restriction enzymes bind to the DNA molecules as homodimers and have the ability to recognize symmetrical DNA sequences as well as asymmetrical sequences.


What is the difference between sticky ends and blunt ends?

Restriction enzymes cut double-stranded DNA in half. Depending on the restriction enzyme, the cut can result in either a sticky end or a blunt end. Sticky ends are more useful in molecular cloning because they ensure that the human DNA fragment is inserted into the plasmid in the right direction.

Similarly, what are blunt ends used for? Blunt ends have no overhang. They cannot match up as specifically as DNA with sticky ends however, they can be useful when sticky ends can't be used. SmaI is a restriction enzyme that makes blunt ends.

Also to know is, what is the difference between blunt ends and sticky ends quizlet?

Sticky ends are when the enzymes make staggered cuts in the two strands. Cuts that are not directly opposite of each other. Sticky ends are most useful in rDNA because they can be used to join two different pieces of DNA that were cut by the same restriction enzyme.

How do you convert blunt ends to sticky ends?

Blunt and sticky ends can be inter converted by either adding restriction cognitive sites or by removing some of them. Blunt end are converted into sticky end prior to cloning.


Restriction Enzymes in DNA: Mode of Action And its Types

Read this article to learn about the restriction enzymes and their mode of action. And it also describes different types of restriction enzymes. The types are: (1) Type I (2) Type II and (3) Type III.

Restriction enzymes are DNA-cutting enzymes found in bacteria (and harvested from them for use). Because they cut within the molecule, they are often called restriction endonucleases.

A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides. For example, the bacterium Hemophilus aegypticus produces an enzyme named Haelll that cuts DNA wherever it encounters the sequence

The cut is made between the adjacent G and C. This particular sequence occurs at 11 places in the circular DNA molecule of the virus phiX174. Thus treatment of this DNA with the enzyme produces 11 fragments, each with a precise length and nucleotide sequence. These fragments can be separated from one another and the sequence of each determined. Haelll and Alul cut straight across the double helix producing “blunt” ends. However, many restriction enzymes cut in an offset fashion.

The ends of the cut have an overhanging piece of single-stranded DNA. These are called “sticky ends” because they are able to form base pairs with any DNA molecule that contains the complementary sticky end (Fig. 13.16). Any other source of DNA treated with the same enzyme will produce such molecules. Mixed together, these molecules can join with each other by the base pairing between their sticky ends.

The union can be made permanent by another enzyme, DNA ligase that forms covalent bonds along the backbone of each strand. The result is a molecule of recombinant DNA (rDNA). Because recognition sequences and cleavage sites differ between restriction enzymes, the length and the exact sequence of a sticky-end “overhang”, as well as whether it is the 5′ end or the 3′ end strand that overhangs, depends on which enzyme produced it. Base-pairing between overhangs with complementary sequences enables two fragments to be joined or “spliced” by a DNA ligase.

A sticky-end fragment can be ligated not only to the fragment from which it was originally cleaved, but also to any other fragment with a compatible sticky end. The sticky end is also called a cohesive end or complementary end in some reference.

If a restriction enzyme has a non- degenerate palindromic (the sequence on one strand reads the same in the same direction on the complementary strand e.g. GTAATG is not a palindromic DNA sequence, but GTATAC is, GTATAC is complementary to CATATG) cleavage site, all ends that it produces are compatible. Ends produced by different enzymes may also be compatible.

Naming:

Restriction enzymes are named based on the bacteria in which they are isolated in the following manner:

I First identified Order ID’d in bacterium

Patterns of DNA Cutting by Restriction Enzymes:

The enzyme cuts asymmetrically within the recognition site such that a short single-stranded segment extends from the 5′ ends. BamHI cuts in this manner.

Again, we see asymmetrical cutting within the recognition site, but the result is a single-stranded overhang from the two 3′ ends. Kpnl cuts in this manner.

Enzymes that cut at precisely opposite sites in the two strands of DNA generate blunt ends without overhangs. Smal is an example of an enzyme that generates blunt ends.

The 5′ or 3′ overhangs generated by enzymes that cut asymmetrically are called sticky ends or cohesive ends, because they will readily stick or anneal with their partner by base pairing.

Mode of Action:

A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA (Table 13.4). The enzyme makes two incisions, one through each of the sugar-phosphate backbones (i.e., each strand) of the double helix without damaging the nitrogenous bases.

The chemical bonds that the enzymes cleave can be reformed by other enzymes known as ligases, so that restriction fragments carved from different chromosomes or genes can be spliced together, provided their ends are complementary.

Many of the procedures of molecular biology and genetic engineering rely on restriction enzymes. The term restriction comes from the fact that these enzymes were discovered in E. coli strains that appeared to be restricting the infection by certain bacteriophages.

Restriction enzymes therefore are believed to be a mechanism evolved by bacteria to resist viral attack and to help in the removal of viral sequences. They are part of what is called the restriction modification system. The 1978 Nobel Prize in Medicine was awarded to Daniel Nathans, Werner Arber and Hamilton Smith for the discovery of restriction endonucleases, leading to the development of recombinant DNA technology.

The first practical use of their work was the manipulation of E. coli bacteria to produce human insulin for diabetics. The ability to produce recombinant DNA molecules has not only revolutionized the study of genetics, but has laid the foundation for much of the biotechnology industry. The availability of human insulin (for diabetics), human factor VIII (for males with hemophilia A), and other proteins used in human therapy all were made possible by recombinant DNA.

Types of Restriction Enzymes:

Restriction enzymes are classified biochemically into three types. These are designated as Type I, Type II, and Type III. A major type of Type II enzymes are sometimes referred to as Type IV enzymes.

Type I and III systems, both the methylase and restriction activities are carried out by a single large enzyme complex. Although these enzymes recognize specific DNA sequences, the sites of actual cleavage are at variable distances from these recognition sites, and can be hundreds of bases away. Both require ATP for their proper function.

Type I restriction enzymes produce DNA cleavage following translocation of the DNA, which makes them important molecular motors. The cleavage of DNA appears to occur after blockage of the translocation activity (often following collision with another translocating Type R-M enzyme, but also due to other factors).

These enzymes read the methylation status of their recognition sequence, compare the methylation status of two adenines within the recognition sequence, and if both adenines are un-methylated (a signal that the DNA is non-host DNA), the enzyme undergoes a conformational switch that turns the enzyme into a molecular motor and endonuclease.

However, if either one of the adenines is methylated (a signal that the DNA is host DNA) then the enzyme acts as a maintenance methylase and methylates the other adenine. In Type II systems, the restriction enzyme is independent of its methylase, and cleavage occurs at very specific sites that are within or close to the recognition sequence. The vast majority of known restriction enzymes are of type II, and it is these that find the most use as laboratory tools. They produce discrete bands during gel electrophoresis, and are useful for DNA analysis and gene cloning. The first to be discovered and utilized was EcoRI, which is staggered and its recognition sequence is 5′-GAATTC-3′.

Type II enzymes are further classified according to their recognition site. Most type II enzymes cut palindromic DNA sequences, while type IIa enzymes recognise non-palindromic sequences and cleave outside of the recognition site, and type lIb enzymes cut sequences twice at both sites outside the recognition sequence. Type lIs enzymes cleave the DNA at a considerable offset from the recognition sequence. The most common type II enzymes are those like HhaI, HindIII and NotI that cleave DNA within their recognition sequences.

Restriction enzymes usually occur in combination with one or two modification enzymes (DNA-methyltransferases) that protect the cell’s own DNA from cleavage by the restriction enzyme.

Modification enzymes recognize the same DNA sequence as the restriction enzyme that they accompany, but instead of cleaving the sequence, they methylate one of the bases in each of the DNA strands. The methyl groups protrude into the major groove of DNA at the binding site and prevent the restriction enzyme from acting upon it.

Together, a restriction enzyme and its “cognate” modification enzyme(s) form a restriction-modification (R-M) system. In some R-M systems the restriction enzyme and the modification enzyme(s) are separate proteins that act independently of each other. In other systems, the two activities occur as separate subunits, or as separate domains, of a larger, combined, restriction-and-modification enzyme.

Restriction Enzymes as Tools:

Recognition sequences typically are only four to twelve nucleotides long. Because there are only so many ways to arrange the four nucleotides—A,C,G and T–into a four or eight or twelve nucleotide sequence, recognition sequences tend to “crop up” by chance in any long sequence. Furthermore, restriction enzymes specific to hundreds of distinct sequences have been identified and synthesized for sale to laboratories.

As a result, potential “restriction sites” appear in almost any gene owr chromosome. Meanwhile, the sequences of some artificial plasmids include a “linker” that contains dozens of restriction enzyme recognition sequences within a very short segment of DNA. So no matter the context in which a gene naturally appears, there is probably a pair of restriction enzymes that can cut it out, and which will produce ends that enable the gene to be spliced into a “plasmid”.

Another use of restriction enzymes can be to find specific SNPs. If a restriction enzyme can be found such that it cuts only one possible allele of a section of DNA (that is, the alternate nucleotide of the SNP causes the restriction site to no longer exist within the section of DNA), this restriction enzyme can be used to genotype the sample without completely sequencing it. The sample is first run in a restriction digest to cut the DNA, and then gel electrophoresis is performed on this digest.

If the sample is homozygous for the common allele, the result will be two bands of DNA, because the cut will have occurred at the restriction site. If the sample is homozygous for the rarer allele, the sample will show only one band, because it will not have been cut. If the sample is heterozygous at that SNP, there will be three bands of DNA. This is an example of restriction mapping.