Science Project Title

Restriction EnzymeBy Ananya Azad Hrisha

OverviewDefinition.Brief Description.Why named so?Nomenclature.Characteristics.Mode of Actions.Types.Impacts & Uses.Use of Restriction Enzymes in Recombinant DNA Technonoly.Restriction Enzyme Recognition Sequences.Summary.

RESTRICTION ENZYME/RESTRICTION ENDONUCLEASEEnzymes that cut DNA at or near specific recognition nucleotide sequences known as restriction sites.Especial class of enzymes that cleave (cut) DNA at a specific unique internal location along its length. Often called restriction endonucleases (Because they cut within the molecule).Discovered in the late 1970s by Werner Arber, Hamilton Smith, and Daniel Nathans.Essential tools for recombinant DNA technology.Naturally produced by bacteria that use them as a defense mechanism against viral infection. Chop up the viral nucleic acids and protect a bacterial cell by hydrolyzing phage DNA.

Summarize your research in three to five points.

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RESTRICTION ENZYMEThe bacterial DNA is protected from digestion because the cell methylates (adds methyl groups to) some of the cytosines in its DNA. The purified forms of these bacterial enzymes are used in today's laboratories.Commonly classified into three types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix.

Nomenclature

Since their discovery in the 1970s, many restriction enzymes have been identified; for example, more than 3500 different Type II restriction enzymes have been characterized. Each enzyme is named after the bacterium from which it was isolated, using a naming system based on bacterial genus, species and strain. For example, the name of the EcoRI restriction enzyme was derived as shown in the box.Derivation of the EcoRI nameAbbreviationMeaningDescriptionEEscherichiagenuscocolispecific epithetRRY13strainIFirst identifiedorder of identificationin the bacterium

CharacteristicsMost restriction enzymes are specific to a single restriction site Restriction sites are recognized no matter where the DNA came from The number of cuts in an organism's DNA made by a particular restriction enzyme is determined by the number of restriction sites specific to that enzyme in that organism's DNA. A fragment of DNA produced by a pair of adjacent cuts is called a RESTRICTION FRAGMENT. A particular restriction enzyme will typically cut an organism's DNA in to many pieces, from several thousand to more than a million! There is a great deal of variation in restriction sites even within a species.

The enzyme "scans" a DNA molecule, looking for a particular sequence, usually of four to six nucleotides. Once it finds this recognition sequence, it stops and cuts the strands. This is known as enzyme digestion. On double stranded DNA the recognition sequence is on both strands, but runs in opposite directions. This allows the enzyme to cut both strands. Sometimes the cut is blunt, sometimes the cut is uneven with dangling nucleotides on one of the two strands. This uneven cut is known as sticky ends.

Mode of Action

Mode of action (how R.E. cuts DNA)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.Restriction enzymes hydrolyze the backbone of DNA between deoxyribose and phosphate groups. This leaves a phosphate group on the 5' ends and a hydroxyl on the 3' ends of both strands. A few restriction enzymes will cleave single stranded DNA, although usually at low efficiency. The restriction enzymes most used in molecular biology labs cut within their recognition sites and generate one of three different types of ends. In the diagrams below, the recognition site is boxed in yellow and the cut sites indicated by red triangles.5' overhangs: 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.

Mode of action (how R.E. cuts DNA)

Blunts: Enzymes that cut at precisely opposite sites in the two strands of DNA generate blunt ends without overhangs. SmaI 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. The sticky end is also called a cohesive end or complementary end in some reference.3' overhangs: Again, we see asymmetrical cutting within the recognition site, but the result is a single-stranded overhang from the two 3' ends. KpnI cuts in this manner.

Kinds of Restriction Enzymes

Star Activity of Restriction EnzymesStar activity is defined as the alteration in the digestion specificity that occurs under sub-optimal enzyme conditions. Star activity results in cleavage of DNA at non-specific sites. Some of the sub-optimal conditions that result in star activity are as follows:pH >8.0glycerol concentration of >5%enzyme concentration >100 units/mg of DNAincreased incubation time with the enzymepresence of organic solvents in the reaction mixtureincorrect cofactor or bufferType VType V restriction enzymes (e.g., the cas9-gRNA complex from CRISPRs) utilize guide RNAs to target specific non-palindromic sequences found on invading organisms. They can cut DNA of variable length, provided that a suitable guide RNA is provided. The flexibility and ease of use of these enzymes make them promising for future genetic engineering applications

Artificial restriction enzymes

Artificial restriction enzymes can be generated by fusing a natural or engineered DNA binding domain to a nuclease domain (often the cleavage domain of the type IIS restriction enzyme FokI).

Such artificial restriction enzymes can target large DNA sites (up to 36 bp) and can be engineered to bind to desired DNA sequences.

Zinc finger nucleases - are the most commonly used artificial restriction enzymes and are generally used in genetic engineering applications, but can also be used for more standard gene cloning applications.

Other artificial restriction enzymes are based on the DNA binding domain of TAL effectors.

CRISPR RNA molecules are also Artificial restriction enzymes.

Restriction Enzyme Recognition Sequences

The length of restriction recognition sites varies: The enzymes EcoRI, SacI and SstI each recognize a 6 base-pair (bp) sequence of DNA, whereas NotI recognizes a sequence 8 bp in length, and the recognition site for Sau3AI is only 4 bp in length. Length of the recognition sequence dictates how frequently the enzyme will cut in a random sequence of DNA. Enzymes with a 6 bp recognition site will cut, on average, every 46 or 4096 bp; a 4 bp recognition site will occur roughly every 256 bp.

Different restriction enzymes can have the same recognition site - such enzymes are called isoschizomers: Look at the recognition sites for SacI and SstI - they are identical. In some cases isoschizomers cut identically within their recognition site, but sometimes they do not. Isoschizomers often have different optimum reaction conditions, stabilities and costs, which may influence the decision of which to purchase.

Restriction Enzyme Recognition Sequences

Restriction recognitions sites can be unambiguous or ambiguous: The enzyme BamHI recognizes the sequence GGATCC and no others - this is what is meant by unambiguous. In contrast, HinfI recognizes a 5 bp sequence starting with GA, ending in TC, and having any base between (in the table, "N" stands for any nucleotide) - HinfI has an ambiguous recognition site. XhoII also has an ambiguous recognition site: Py stands for pyrimidine (T or C) and Pu for purine (A or G), so XhoII will recognize and cut sequences of AGATCT, AGATCC, GGATCT and GGATCC.

The recognition site for one enzyme may contain the restriction site for another: For example, note that a BamHI recognition site contains the recognition site for Sau3AI. Consequently, all BamHI sites will cut with Sau3AI. Similarly, one of the four possible XhoII sites will also be a recognition site for BamHI and all four will cut with Sau3AI. Other point to notice from the table above is that most recognition sequences are palindromes - they read the same forward (5' to 3' on the top strand) and backward (5' to 3' on the bottom strand). Most, but certainly not all recognition sites for commonly-used restriction enzymes are palindromes. Most restriction enzymes bind to their recognition site as dimers (pairs), as depicted for the enzyme PvuII in the figure to the right.

THE IMPACT OF RESTRICTION ENZYMES

Genetic engineering

Type II enzymes yielded many practical benefits, as E. coli K12, its genes and its vectors became the workhorses of molecular biology in the 1970s for cloning, generation of libraries, DNA sequencing, detection and overproduction of enzymes, hormones, etc.

The applications of Type II enzymes continued to expand, especially after the arrival of synthetic DNA, in vitro packaging of DNA in phage particles and improved bacterial hosts and vectors for overexpression and stabilization of proteins.

Production of insulin from recombinant bacteria and yeast by Genentech, thus greatly increasing the supply for diabetics and the production of a recombinant vaccine for Hepatitis B by Biogen to treat the hundreds of millions of people at risk of infection by this virus.

Zinc-finger nucleases and the TAL-effector nucleases, which have potential for gene targeting and gene therapy.

THE IMPACT OF RESTRICTION ENZYMES

Restriction enzymes are tools for monitoring Restriction Fragment Length Polymorphisms (RFLP), allowing the location of mutations, generation of human linkage maps, identification of disease genes (such as sickle cell trait or Huntington disease), and last, but not least, the DNA fingerprinting technique developed by Alec Jeffreys.

DNA fingerprinting

DNA fingerprinting allows the solution of paternity cases, the identification of criminals and their victims and the exoneration of the falsely accused. The use of REases in this system enabled the creation of suitable procedures for such identification.

Useful for identifying pathogenic bacterial strains, most recently of S. aureus sp with antibiotic-resistance and virulence factors mediated by mobile genetic elements, e.g. the methicillin-resistant S. aureus (MRSA) bacteria.

Why restriction enzymes are important for rDNA techniques?

Restriction enzyme recognizes and cuts, or digests, only one particular sequence of nucleotide bases in DNA

Typical restriction enzymes used in cloning experiments recognize four-, six-, or eight-base sequences.

It cuts this sequence in the same way each time.

Hundreds of restriction enzymes are known, each producing DNA fragments with characteristic ends.

The role of a restriction enzyme in making recombinant DNA

Conclusion

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