Restriction Enzymes What Are Restriction Enzymes? • Enzymes: What Are Restriction Enzymes? • Recognize: • Naturally found • Important tool Discovery of Restriction Enzymes First restriction enzyme: Werner Arber Daniel Nathans Hamilton Smith Werner Arber – discovered restriction 1978 Nobel prize for Medicine and enzymes Physiology was presented for Daniel Nathans - pioneered the application discovery of restriction enzymes.) of restriction for the construction of genetic maps Hamilton Smith - showed that restriction enzyme cuts DNA in the middle of a specific sequence Biological Role of Restriction Enzymes • Naturally found in different types of bacteria. • Protection Biological Role of Restriction Enzymes • Why don’t bacteria destroy their own DNA with their restriction enzymes? Restriction-Modification System of Bacteria Restriction Endonucleases (enzymes that cleave DNA at specific sites) Foreign DNA Host DNA Part I: Restriction Part II: Modification Bacteria produce restriction Bacteria methylate their DNA to enzymes that digest foreign (viral DNA) protect it from digestion How Many Restriction Enzymes Are There? • 10,000 bacteria species • 3000 restriction enzymes • 600 are available commercially Nomenclature of Restriction Enzymes • Each enzyme is named after the bacterium from which it was isolated using a naming system based on bacterial genus, species, and strain. Nomenclature of Restriction Enzymes • Example: EcoR1 • Genus: • Species: • Strain: • Order discovered: Examples of Restriction Enzymes Recognized Enzyme Organism source Sequence 5' GAATTC 3’ EcoRI Escherichia coli 3' CTTAAG 5’ 5' TCGA 3’ TaqI Thermus aquaticus 3' AGCT 5’ 5'AAGCTT 3’ HindIII Haemophilus influenzae 3'TTCGAA 5’ 5' GGATCC 3’ BamHI Bacillus amyloliquefaciens 3' CCTAGG 5’ 5' AGCT 3’ AluI Arthrobacter luteus 3' TCGA 5’ What Sequences Do Restriction Enzymes Recognize? • Known as restriction / cleavage sites • “Able was I, ere, I saw Elba”; Mom; Dad??? What Sequences Do Restriction Enzymes Recognize? • DNA Palindromes: • Mirror-like palindrome DNA Palindromes: The EcoRI cutting site: 5'-GAATTC-3' • Inverted repeat palindrome 3'-CTTAAG-5' The HindIII cutting site: 5'-AAGCTT-3' 3'-TTCGAA-5' Enzyme Site Recognition Restriction site Palindrome • Each enzyme digests (cuts) DNA at a specific sequence = restriction site • Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC) Fragment 1 Fragment 2 Types of Restriction Digest Ends • When restriction enzymes cut, they produce either: 5’ G A A T T C 3’ 5’ G A T A T C 3’ 3’ C T T A A G 5’ 3’ C T A T A G 5’ Sticky Ends Blunt Ends Blunt vs Sticky Ends • Blunt Ends • Sticky Ends Types of Restriction Digest Ends Types of Restriction Enzymes • Restriction endonucleases are categorized into three general groups. • Based on: Types of Restriction Enzymes • Restriction endonucleases are categorized into three general groups. • Type I Types of Restriction Enzymes • Restriction endonucleases are categorized into three general groups. • Type II Types of Restriction Enzymes • Type II Restriction Enzymes are the Most Useful for Cloning Examples of Type II Restriction Endonucleases Blunt ends Cohesive “sticky” ends Types of Restriction Enzymes • Restriction endonucleases are categorized into three general groups. • Type III Uses of Restriction Enzymes • Recombinant DNA technology Restriction Enzymes for Transformation Human DNA cleaved with EcoRI Corn DNA cleaved with EcoRI PO -A-A-T-T-C-A-G-C-T-A-C-G-3’ 5’-C-G-G-T-A-C-T-A-G-OH + 4 3’-G-C-C-A-T-G-A-T-C-T-T-A-A-PO4 HO-G-T-C-G-A-T-G-C-5’ Complementary base pairing 5’-A-C-G-G-T-A-C-T-A-G A-A-T-T-C-A-G-C-T-A-C-G-3’ 3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A G-T-C-G-A-T-G-C-5’ + DNA Ligase, + rATP 5’-A-C-G-G-T-A-C-T-A-G-A-A-T-T-C-A-G-C-T-A-C-G-3’ 3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A-G-T-C-G-A-T-G-C-5’ recombinant DNA molecule Uses of Restriction Enzymes • We can use restriction enzymes to find out information about a piece of DNA: Uses of Restriction Enzymes • Restriction Enzymes can be used to generate a restriction map. Uses of Restriction Enzymes • Restriction Enzymes can be used to generate a restriction map. • Provides useful information in characterizing a DNA molecule. Restriction Maps • A map showing positions of restriction sites in a DNA sequence Restriction Digest • Cutting DNA at each restriction site creates multiple restriction fragments: • Is it possible to reconstruct the order of the fragments from the sizes of the fragments {3,5,5,9} ? Restriction Digest • Alternative ordering of restriction fragments: vs. Restriction Digestion Rxn • The activity of restriction enzymes is dependent upon precise environmental conditions: Restriction Digestion Rxn • The activity of restriction enzymes is dependent upon precise environmental conditions: • Why incubate at 37°C? • What happens if the temperature is too hot or cool? Restriction Digest Prediction • By choosing correctly, A four-base cutter: enzymes can cut DNA very (Sau3A) GATC precisely. • Q: How Frequently Will a Restriction Enzyme Cut DNA? A six-base cutter: • A: (BamHI) GGATCC Discovery of Restriction Enzymes • 1952-53: • 1962: Discovery of Restriction Enzymes • 1970: • First restriction enzyme: • 1978 Nobel prize for Medicine and Physiology was presented for discovery of restriction enzymes. Biological Role of Restriction Enzymes • Why don’t bacteria destroy their own DNA with their restriction enzymes? • Bacteria have mechanisms to protect themselves from the actions of their own restriction enzymes What Sequences Do Restriction Enzymes Recognize? • Known as restriction / cleavage sites • “Able was I, ere, I saw Elba” What Sequences Do Restriction Enzymes Recognize? • DNA Palindromes: • Mirror-like palindrome • Inverted repeat palindrome Uses of Restriction Enzymes • Recombinant DNA technology • Gene cloning and protein expression (transformation). Restriction Digestion Rxn • The activity of restriction enzymes is dependent upon precise environmental conditions: • Restriction Buffer provides optimal conditions.