Recombinant DNA: Restriction Enzymes

• Lecture is about recombinant DNA technology, focusing on restriction enzymes (REs).

Learning Objectives

• Describe restriction endonucleases
• Understand the biological role of restriction endonucleases
• Understand the nomenclature and classification of restriction endonucleases
• Describe their general mode of action/operation
• Understand the importance of RE's to R-DNA Technology

Restriction Endonucleases

• Restriction endonucleases, also known as restriction enzymes, are 'molecular scissors'.
• They were discovered in bacteria in 1962.
• They are capable of specifically binding to and cleaving double-stranded DNA (dsDNA).
• E. coli bacteria utilize restriction endonucleases as an enzymatic immune system to recognize and destroy foreign DNA.
• Approximately 3,000 enzymes have so far been identified; and around 200 have been found to have unique properties.
• Hundreds of enzymes have been purified and are commercially available.

DNA Degradation

• DNA degradation occurs via nucleases.
• DNA nucleases (DNases) and RNA nucleases (RNases) exist.
• Endonucleases cut within single-stranded or double-stranded DNA fragments.
• Exonucleases cut at the ends of single-stranded fragments in a direction-specific manner.

Restriction Enzymes in Bacteria

• E. coli bacteria use restriction enzymes as an enzymatic immune system to recognize and destroy foreign DNA.
• Bacteria do not destroy their own DNA with their restriction enzymes.
• The specific DNA binding site of a RE is the recognition sequence.
• The specific cleavage point of a RE is known as a restriction site.

Host DNA Protection

• Bacteria protect their own DNA by modifying their recognition sequences.
• DNA methyltransferases or methylases in the RE add a methyl group (CH3) to specific bacterial DNA nucleotides.
• REs will not cleave at methylated restriction sites on host DNA.
• Invading DNA, which is usually not methylated, will be cleaved and inactivated.

Types of Restriction Endonucleases

• REs are categorized into three groups based on:
  • Composition
  • Enzyme cofactor requirements
  • Nature of target sequence
  • Position of DNA cleavage site relative to target sequence
• Type II REs are the most commonly used in Molecular Biology because:
  • Restriction activity and modifying activity are in separate enzymes
  • Cleave within the recognition site or adjacent to it
  • Do not need ATP for activity - only need Mg2+
  • Cuts in a precise manner, very predictable
  • Have short palindromic recognition sequence
• Type II is optimal for Rec DNA technology.
• It is most useful for gene analysis and cloning.
• There are more than 3500 REs
• They recognize 4-6 bp sequences.
• Needs Mg2+ as cofactor.
• Cut in close proximity of the recognition site
• Homodimers
• ATP hydrolysis is not required

Recognition and Restriction Sites

• REs recognize and cut more than 100 different recognition sequences at a restriction site.
• Most recognition sequences are 4 to 6 bases long.
• Palindromic sequences read the same forward and backward (symmetry) e.g. RADAR, Madam I'm Adam.
• Hindlll is an H. influenzae RE that recognizes the following recognition sequence: 5'-AAGCTT-3' and 3'-TTCGAA-5'.

Nomenclature of REs

• The first three letters of a restriction enzyme's name are abbreviations of the bacterial GENUS and SPECIES from which the enzyme has been isolated, e.g., Eco for E. coli and Hin for H. influenzae.
• The fourth letter represents the particular bacterial STRAIN, e.g., BamH for B. amyloliquefaciens Strain H.
• Roman numerals denote ORDER of discovery from the same strain, e.g., EcoRI - E. coli Strain R - 1st enzyme characterized.
• In cases where the restriction and modification systems are genetically specified by a virus or plasmid, the extra chromosomal element is identified by Arabic numerals, e.g., EcoP1l has methyltransferase abilities from bacteriophage P1.

Mode of action

• Scanning
• Recognition
• Cleavage

Classification of RES

• REs are classified based on cleavage at recognition sequences.
• Types include:
  • Blunt end vs. Cohesive end
  • Rare vs. Frequent
  • Ambiguous vs. Precise
  • Isoschizomers

Blunt vs. Cohesive Ends

• Blunt ends are formed by RE's that cut straight across the double helix (cut DNA at opposite base).
• Cohesive ends are formed by RE's that cut in an offset (staggered) fashion to produce a 5' overhang of single-stranded DNA (ssDNA).
• Cohesive or "sticky" ends can bind to other ssDNA by complementary base pairing.

Rare vs Frequent Cutters

• There are 4 different bases, so the probability of finding a particular base at one location on a DNA strand = 1/4.
• The probability of finding the required base at each of n locations = (1/4)^n, where n = length of recognition seq
• Frequency of sites = 4^n
  • Recognition sequence of n bp has a cleavage frequency  of (1/4)^n
  • 4 bp (Alul, Hpall, Hhal) cleavage frequency is (1/4)^4 = 1/256
  • 6 bp (BamHI, HindIII) has (1/4)^6 = 1/4,096
  • 8 bp (Notl) has (1/4)^8 = 1/65,536
  • 9 bp (AlwNI) has (1/4)^9 = 1/262,144

Ambiguous vs Precise Cutters

• An example of an Ambiguous cuter is BSiE1.

Isoschizomers

• RE pairs that have the same target site (recognition seq)
• Two subtypes:
  • Different cleavage sites (neoschizomers)
    • e.g. Smal and Xmal
  • Same cleavage sites (true isoschizomers)
    • e.g. Aatl and Stul
• There are some REs that have different target sites and produce compatible cohesive ends e.g., BamH1, Bgl3, Mbo1, Sau3A1, BstY1 (isocaudamers).

Applications of Restriction Endonucleases

• The discover of enzymes that cut and paste DNA made genetic engineering possible.
• Restriction enzymes cut DNA and generate fragments.
• Genomic DNA is too large to analyze as is, but smaller fragments are easier to work with.
• Ligase joins different DNA fragments.
• DNA fragments from different species can be ligated (joined) to create Recombinant DNA

Molecular Cloning

• The basic process of cloning a gene (from a chromosome) involves isolating a gene of interest via PCR or Restriction digest.
• Restriction enzymes, Ligase DNA, RE, and DNA play important roles in the cloning strategy.

Restriction Digestion - Step 1

• Reagents and volumes needed:
  • Sterile, deionized water: 16.3 µl
  • RE 10X Buffer: 2.0 µl
  • Acetylated BSA (10µg/µl): 0.2 µl
  • DNA (1µg/µl): 1.0
  • Restriction enzyme (10u/µl): 0.5 µl
  • FINAL VOLUME: 20.0 - Mix by pipetting then add. -Mix by pipetting

Commercial Restriction Enzymes

• Available commercially at 5 units per µL concentration
• 1 unit = 1 unit (U) is the amount of enzyme that catalyzes the reaction of 1 nmol of substrate per minute under standard conditions
• Enzymes come in storage buffer containing EDTA, DTT, BSA and Glycerol
• Too much enzyme in the reaction mix can cause star activity

Star Activity

• Loss of specificity of the restriction enzyme (Type II) under extreme non-standard conditions
• Under such conditions, the restriction enzyme may cleave similar but non identical target sequences
• Non-standard conditions include

1. High glycerol concentration >5% v/v

2. 100 units per ug of DNA

3. Low ionic strength buffer <25mM (usually is 100-150 mM)
4. High pH (> 8.0) usually pH is around 7
5. Presence of organic solvents, DMSO, ethanol, PEG, dimethyl acetamide
6. Cations other than Mg2+ e.g. Cu2+, Co2+, Zn2+, Mn2+

Stopping the Digest

• Heat inactivation of restriction enzymes: Place reaction mix in a water-bath set at 65°C, note that not all restriction enzymes can be heat-inactivated, e.g. Pvul, BgIII, BamHI, Accl.
• Add 2ul of 25 mM EDTA: EDTA chelates the Mg2+ and makes it unavailable to the RE.

Gel Electrophoresis - Step 2

• Gel Electrophoresis of restriction digest: the DNA is negatively charged from the phosphate backbone
• Visualize DNA with ethidium bromide: fluoresces ONLY when bound to DNA.
• Odd wells contain undigested HBB gene 364 bp
• Even wells contain HBB gene fragments following Ddel digestion: -4bp- 1/256 -6bp- 1/4096 -8bp 1/65,536 -9bp- 1/262,144

Single and Double Digestion

• Number and Fragment size differs from enzyme to enzyme

Mutations Affect Digestion

• A single basepair change can "destroy" the recognition site for the Restriction Enzyme.
• This would give a different digest pattern for the same stretch of DNA.
• If occurring in one species vs another, this can be used as a marker to differentiate between them.