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RESTRICTION ENZYMES ( NUCLEASES LIGASE )

Restriction enzymes are proteins that recognize specific nucleotide sequences in DNA and make precise cuts at those sites, primarily found in bacteria to protect against viruses. These enzymes are crucial in recombinant DNA technology for gene analysis and manipulation, creating either blunt or 'sticky' ends, allowing for the joining of DNA fragments. Additionally, there are various types of restriction enzymes, classified by their cutting patterns and specificities, each serving distinct roles in genetic research and biotechnological applications.

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•A restriction enzyme is a protein that recognizes a specific, short nucleotide sequence and cuts the DNA only at that specific site, which is known as restriction site or target sequence.
•Restriction enzymes are found in bacteria (and other prokaryotes). They recognize and bind to specific sequences of DNA, called restriction sites. •Each restriction enzyme recognizes just one or a few restriction sites
•When it finds its target sequence, a restriction enzyme will make a double-stranded cut in the DNA molecule. •These enzymes, which can be purified from bacteria, cut the DNA double helix at specific sites defined by the local nucleotide sequence, thereby cleaving a long double-stranded DNA molecule into fragments of strictly defined sizes. •Typically, the cut is at or near the restriction site and occurs in a tidy, predictable pattern.
•Different species of bacteria make different restriction nucleases, which protect them from viruses by degrading incoming viral DNA. •Each nuclease recognizes a specific sequence of four to eight nucleotides in DNA. These sequences, where they occur in the genome of the bacterium itself, are protected from cleavage by methylation at an A or a C residue
•the sequences in foreign DNA are generally not methylated and so are cleaved by the restriction nucleases. •Large numbers of restriction nucleases have been purified from various species of bacteria; several hundred, most of which recognize different nucleotide sequences, are now available commercially.
The DNA nucleotide sequences recognized by four widely used restriction nucleases such sequences are often six base pairs long and “palindromic” (that is, the nucleotide sequence is the same if the helix is turned by 180 degrees around the center of the short region of helix that is recognized) The enzymes cut the two strands of DNA at or near the recognition sequence.
•For some enzymes, such as HpaI, the cleavage leaves blunt ends • EcoRI, HindIII, and PstI, the cleavage is staggered and creates cohesive ends. • Restriction nucleases are obtained from various species of bacteria: HpaI is from Hemophilus parainfluenzae, EcoRI is from Escherichia coli, HindIII is from Hemophilus influenzae, and PstI is from Providencia stuartii.
•Some restriction nucleases produce staggered cuts, which leave short single-stranded tails at the two ends of each fragment. • Ends of this type are known as cohesive ends, as each tail can form complementary base pairs with the tail at any other end produced by the same enzyme
How Do Restriction Enzymes Cut DNA Sequences? •Restriction enzymes dismantle foreign DNA by cutting it into fragments. This disassembling process is called restriction. •Recombinant DNA technology relies on restriction enzymes to produce new combinations of genes. •The cell protects its own DNA from disassembly by adding methyl groups in a process called modification. •DNA ligase is a very important enzyme that helps to join DNA strands together via covalent bonds.
•In a typical cell, methyl groups (CH3 ) are added to the bases in the sequence to prevent recognition by the restriction enzymes. • This process is carried out by complementary enzymes that recognize the same sequence of nucleotide bases as restriction enzymes.
•The methylation of DNA is known as modification. •With the processes of modification and restriction, cells can both cut up foreign DNA that pose a danger to the cell while preserving the important DNA of the cell.
•The 5' end has a phosphate group attached while the other 3' end has a hydroxyl group attached. •Since the DNA is cut on both strands, there will be complementary ends that can hydrogen bond to one another. These ends are often called "sticky ends."
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 hydrogen bond and able to hold the different fragments together.
•Restriction Enzyme Types : •There are four broad categories of restriction enzymes: Type I enzymes, Type II enzymes, Type III enzymes, and Type IV enzymes •All have the same basic function, but the different types are classified based on their subunit composition, cleavage position, sequence specificity and cofactor requirements.
•Type I enzymes cut DNA at locations distant to the recognition sequence •they have little practical value since they do not produce discrete restriction fragments or distinct gel-banding patterns.
•Type II cut DNA within or close to the recognition sequence • They produce discrete restriction fragments and distinct gel banding patterns, and they are the only class used in the laboratory for routine DNA analysis and gene cloning.
•Type II enzymes frequently differ so completely in amino acid sequence from one another, and indeed from every other known protein, that they exemplify the class of rapidly evolving proteins that are often indicative of involvement in host-parasite interactions. •The most common Type II enzymes are those like HhaI , HindIII and NotI , that cleave DNA within their recognition sequences
•Type III cut DNA near recognition sequences •They cleave outside of their recognition sequences and require two such sequences in opposite orientations within the same DNA molecule to accomplish cleavage; they rarely give complete digests. • Type IV cleave methylated DNA. exemplified by the McrBC and Mrr systems of E. coli.
Products of Restriction Digestion Restriction digestion of double-stranded DNA produces two kinds of ends: Sticky ends and Blunt ends. Blunt ends possess a 5’-phosphate group that promotes ligation. They are universally compatible with other blunt-ended DNA. Blunt ends generated by EcoR V
•The digestion product of DNA strands may result in a fragment with either blunt ends or cohesive (sticky) ends at both the ends. •Cohesive 5' ends generated by EcoR I
•Sticky ends are small stretches of single-stranded DNA capable of self-ligation or ligation with a complementary region from another DNA molecule. The sticky ends possess 3’- or 5’-overhangs of 1–4 nucleotides. •5’ Cohesive end generated by Bln I Bln I (Avr II) from Brevibacterium linens
•3’ Cohesive end generated by Kpn I • Kpn I from Klebsiella pneumoniae
Factors affecting the activity of restriction enzymes •Depending on the substrate DNA and the reaction conditions, restriction enzymes show a wide variation of cleavage and possible star activity. In order to obtain the desired cleavage, it becomes important to control the following factors: •Star activity: Under sub-optimal reaction conditions, some restriction enzymes cleave base sequences at sites different from the defined recognition sequence. In other words, they cleave at non-specific sites. • This phenomenon is called star activity. Some of the factors that induce star activity are high salt and glycerol concentration, presence of impurities, excessive enzyme compared to substrate DNA, increased incubation time, or incompatible buffer and cofactor.
•Methylated DNA: Several DNA molecules are methylated at the recognition site, making them resistant to cleavage by certain restriction enzymes. •Temperature: Most endonucleases optimally digest the target DNA at 37 °C. However, there are some exceptions with lower or higher optimal temperatures. For example, Taq I optimally digests at 65 °C and Apa I digests at 25 °C.
•The first major application was as a tool for cutting DNA into fragments in ways that would make it easier to study and, in particular, identify and characterize genes. • A second major use was as a device for recombining, or joining, DNA molecules from different genomes, usually with the goal of identifying and characterizing a gene or studying gene expression and regulation (Heinrichs, 2007).