Molecular Biology: Nucleases & Restriction Enzymes PDF

Molecular Biology Dr Nzar Shwan MLT department Lecture 09: Monday, 2nd December, 2023 4th Year (Semester 7) Nucleases: Restriction Enzymes (Restriction Endonucleases) Nucleases: Nucleases are enzymes that degrade DNA...

Molecular Biology Dr Nzar Shwan MLT department Lecture 09: Monday, 2nd December, 2023 4th Year (Semester 7) Nucleases: Restriction Enzymes (Restriction Endonucleases) Nucleases: Nucleases are enzymes that degrade DNA molecules by breaking the phosphodiester bonds that link one nucleotide to the next in a DNA strand. These enzymes are involved in various cellular processes, including DNA replication, repair, recombination, and RNA processing. There are two different kinds of nuclease: (1) Exonucleases catalyses hydrolysis of terminal nucleotides from the end of DNA or RNA molecule either 5’to 3’ direction or 3’ to 5’ direction. (2) Endonucleases can recognize specific base sequence (restriction site) within DNA or RNA molecule and cleave internal phosphodiester bonds within a DNA molecule. 1 Restriction Endonuclease (restriction Enzyme) A restriction enzyme is a nuclease enzyme that cleaves DNA sequence at a random or specific recognition sites known as restriction sites. In 1970 the first restriction endonuclease enzyme HindII was isolated. For the subsequent discovery and characterization of numerous restriction endonucleases, in 1978 Daniel Nathans, Werner Arber, and Hamilton O. Smith awarded for Nobel Prize for Physiology or Medicine. Since then, restriction enzymes have been used as an essential tool in recombinant DNA technology. Origin of Restriction Enzymes Restriction enzymes are made naturally by many different species of bacteria and protect bacterial cells from invasion by foreign DNA, particularly that of bacteriophages. Perhaps all, species of bacteria: over 2500 different ones have been isolated and more than 300 are available for use in the laboratory. 2  Restriction Endonuclease Nomenclature: Restriction endonucleases are named according to the organism in which they were discovered, using a system of letters and numbers. For example, HindIII (pronounced “hindee-three”) was discovered in Haemophilus influenza (strain d). The Roman numerals are used to identify specific enzymes from bacteria that contain multiple restriction enzymes indicating the order in which restriction enzymes were discovered in a particular strain. Recognition Sequences: Each restriction enzyme recognizes a specific DNA sequence, which is usually a short, palindromic sequence of nucleotides. Palindromic means that the sequence reads the same forward and backward on complementary strands. For example, the sequence 5'-GAATTC-3' is palindromic. 3 Cutting DNA: Restriction enzymes cleave the DNA at specific points within or near their recognition sequences. There are two types of cuts: a) Blunt Ends: The enzyme cuts the DNA at the same position on both strands, producing blunt ends with no overhanging sequences. b) Sticky Ends: The enzyme cuts the DNA, leaving overhanging single-stranded sequences at the ends. These overhangs are complementary and can base-pair with each other. Types of Restriction Enzymes Three different classes of restriction endonuclease are recognized, each distinguished by a slightly different mode of action. Types I and III are rather complex and have only a limited role in genetic engineering. Types I Cleavage occurs approximately 1000 bp away from the recognition site. Type III restriction enzymes These enzymes recognize and methylate the same DNA sequence but cleave 24–26 bp away. 4  Type II restriction endonucleases, on the other hand, are the cutting enzymes that are so important in gene cloning. Type II restriction enzymes Cleavage of nucleotide sequence occurs at the restriction site. Usually recognize sequences that are palindromic; that is, the sequence in one strand is identical when read in the opposite direction in the complementary strand. For example, the sequence recognized by EcoRI is 5ʹ– GAATTC–3ʹ in the top strand. Read in the opposite direction in the bottom strand, this sequence is also 3ʹ– CTTAAG–5ʹ. The restriction binding site may either be interrupted (e.g., BstEII recognizes the sequence 5´-GGTNACC-3´, where N can be any nucleotide) or continuous (e.g., KpnI recognizes the sequence 5´-GGTACC-3´). Type II endonucleases are widely used for mapping and reconstructing DNA in vitro because they recognize specific sites and cleave just at these sites. 5  Currently, several hundred different restriction enzymes from various bacterial species have been identified and are available commercially to molecular biologists. The following table gives a few examples. Biotechnological Applications of Restriction enzymes: Genetic Engineering: Restriction enzymes are widely used in genetic engineering to cut DNA at specific sites, allowing the insertion of foreign DNA into a vector (such as a plasmid) for cloning purposes. DNA Fragment Analysis: They are used to analyse DNA fragments in techniques like restriction fragment length polymorphism (RFLP) analysis. DNA Sequencing: Some restriction enzymes are used in DNA sequencing methods. 6