Lecture 2 and 3 - Restriction Enzymes

  • A phenomenon called host-controlled variation was described by various groups: Luria and Human (1952), Anderson and Felix (1952) and Bertani and Weigle (1953)

  • Bacteriophages varied in their ability to grow on different host strains

  • Once growth was achieved on one strain, the bacteriophages could continue to grow on this strain but were restricted in their ability to grow on other strains

The finding of host-controlled variation provided the first evidence that:

  • Bacteria applied an identification (modification) tag to their DNA

  • DNA lacking this tag (upon transfer from one strain to another) was recognised as ‘non-self’ and restricted

  • In the 1960s, Werner Arber and Daisy Dussoix demonstrated that the phage DNA carried the host range imprint

  • The imprint was conferred by methylation of DNA and host-controlled variation came to be known as bacterial restriction-modification system

Bacterial restriction-modification system

  • Restriction enzymes protect bacteria from infection by viruses

  • Non-host DNA is cleaved whilst the cell’s own DNA is protected from digestion by site specific methylation by DNA methyltransferases

  • Restriction enzymes occur in combination with DNA methyltransferases, both recognising the same DNA sequence

  • Methylated DNA poses a hinderance to restriction

In 1971, Kathleen Danna and Daniel Nathans demonstrated that:

  • Endonuclease R (discovered by Hamilton Smith and Kent Wilcox) could be used to produce specific fragments of simian virus 40

  • Electrophoresis provided a good tool to separate the DNA fragments

  • In 1978, Arber, Smith and Nathans were awarded the Nobel Prize

Restriction enzymes are needed as naturally occurring DNA molecules (chromosomes and some plasmids) are too large to be manageable in the laboratory eg.

  • Human genome → 3038 Mb

  • Escherichia coli chromosome → 4.64 Mb

  • Streptomyces coelicolor chromosome → 8.6 Mb

What are the properties of restriction enzymes?

  • Hydrolyse phosphodiester bonds in sugar-phosphate backbone of each strand of DNA at specific nucleotide sequences

  • Generate discrete, gene size DNA fragments suitable for manipulation

  • The fragments generated can be re-joined in the lab

Mechanism of action

  • All restriction enzymes catalyse the hydrolysis of DNA phosphodiester bonds, leaving a phosphoryl group attached to the 5’ end and a 3’ hydroxyl group

  • Cleavage takes place by in-line displacement of 3′ oxygen from phosphorus by magnesium-activated water

Four types of restriction enzymes

  • Type I → multisubunit, restriction-modification enzymes (dual activity) that randomly cut DNA away from recognition sequence

  • Type II → cut DNA at defined positions close to or within the recognition sequence, recognise symmetric DNA sequences or palindromes (sequence in 5’-3’ direction on one strand same in the 5’-3’ direction on the other)

  • Type III → large, combination restriction and modification enzymes, recognise specific 5-7 bp sequences and cut DNA outside the recognition sequence

  • Type IV → methylation-dependant restriction enzymes, cleave at variable distance from recognition site

Type I 

  • Identified in E.coli as barriers to foreign DNA entry and have several sub-families (A-E)

  • Present in almost one-half of Bacteria and Archaea

  • Consist of three different subunits

HsdM → modification

HsdR → restriction

HsdS → sequence recognition

Mechanism of Type I

  • Require ATP, Mg2+ and S-adenosylmethionine for activity

  • They interact in general with two asymmetrical bi-partite recognition sites → AACNNNNNNGTGC

  • Translocate the DNA in an ATP-hydrolysis dependent manner and cut the DNA distal to the recognition sites, approximately half-way between two sites

Specific plasticity

  • HsdS subunits of Type I enzymes are responsible for DNA sequences recognition

  • Duplicated organisation comprising of two target recognition domains (TRDs) in tandem

  • Each TDR specifies recognition of one half of the recognition sequence independently 

  • Genetic rearrangements between hsdS genes results in hybrid HsdS subunits with novel specificity

Type III 

  • Recognise two sequences in opposite orientations within same DNA and cleave 25-27 nucleotides away from their recognition site

  • Examples are EcoP1I and EcoP15I

  • Found in most sequenced bacterial genomes

  • Require ATP, Mg2+ and S-adenosylmethionine as cofactors although they do not necessarily need S-adenosylmethionine

  • Number of nucleotides between two sequences is variable

Type III cleavage mechanism

Type III restriction enzymes consist of two subunits:

  • Mod (homodimeric) → responsible for DNA recognition and methylation

  • Res → responsible for ATP hydrolysis, DNA translocation and cleavage

  • Form a hetero-tetrameric Res2Mod2 complex

Subunit composition

  • Type III restriction enzymes comprise two

modification (Mod) subunits, each containing a target

recognition domain (TRD) to bind to the target sequence

  • A MTase catalytic domain to monitor the methylation

    status of an adenine in the target and transfer –CH3 group

    to N6 of adenine

  • And two restriction (Res) subunits each

    containing a DNA helicase and ATP-hydrolysing domain,

    and an endonuclease domain (6, 8)

Type II 

  • Cleaves specifically within or close to its recognition site

  • No requirement of ATP or GTP for its activity

  • Form homodimers of 2 × 30 kDa molecular mass

  • Recognises palindromic sequences of 4-8 bp in length

  • Requires Mg2+ for its activity

  • Cleaves the bond between the 3’-OH and the 5’-phosphate in DNA

  • Classified based on enzymatic behaviour rather than evolutionary relatedness

BamHI EcoRV

Type II subtypes

Target Site Location

  • Binds to DNA non-specifically and then slides along DNA randomly until it encounters the target sequence

Star Activity

  • Restriction enzymes are capable of cleaving sequences which are similar, but not identical, to their defined recognition sequence

  • This altered specificity has been termed star activity

  • For example, EcoRI’s recognition sequence is GAATTC and has star activity at N/AATTN (GGATTT, AAATTT, GAATTT and GAATTA)

What are the causes of star activity?

  • Low ionic strength and alkaline pH

  • Presence of organic solvents, such as glycerol or DMSO

  • When Mg2+ is replaced by Mn2+

  • High enzyme concentrations under optimum buffer conditions

Among the most accurate enzymes known:

  • Cleavage of non-cognate substrates is at least five orders of  magnitude slower than cleavage of the cognate substrate

Different types of DNA ends after restriction

  • 5’overhang ends

  • Blunt ends

  • 3’overhang ends

Why is it important to have different DNA ends?

  • Blunt ends are not sequence-specific and can be joined to any other blunt-ended DNA fragment, regardless of its sequence

  • Sticky ends are useful for cloning because the complementary overhangs can temporarily hold two different DNA fragments together, allowing DNA ligase to permanently join them

Nomenclature of Restriction Enzymes

  • The name reflects origin → species name of the host organism where the restriction enzyme was isolated from

  • The number written as Roman numeral indicates the order in which they were isolated

  • A restriction enzyme will always cut at a specific nucleotide sequence no matter the source of the DNA

  • Recognition sequences can be continuous or discontinuous

  • Continuous sequences → the bases are all adjacent and the enzyme cuts within this sequence eg. Kpnl 5’-GGTACC-3’

  • Discontinuous sequences → the recognised bases are separated by one or more non-specific (spacer) nucleotides and enzyme identifies two short half-sites eg. Xmnl 5’GAANNNNTTC-3’

  • Recognition sequences can vary in length eg. EcoRI 5’GAATTC-3’ and Notl 5’-GCGGCCGC-3’

  • Isoschizomers can recognise the same recognition site but cut at a different position

DNA ligases

  • Catalyse the formation of phosphodiester bond between two DNA chains that are part of a double helix

  • Requires a free hydroxyl group (OH) at the 3’ end of one DNA chain and a phosphate group (Pho) at the 5’ end of the other 

  • Can ligate ‘sticky’ or blunt ends

  • Their natural role is to seal nicks in the backbone of double-stranded DNA after replication, recombination or repair processes

  • Energy sources for Ligase activity: 

 Escherichia coli → NAD+

Animal cells and bacteriophages → ATP

  • T4 DNA Ligase from bacteriophage T4 is the most commonly used in the lab

DNA ligase mechanism of action

  1. Ligase reacts with ATP to form a covalent enzyme-ADP complex

  2. Ligase-AMP complex activates phosphate group at 5’end of DNA

  3. Nucleophilic attack by OH group at 3’end of DNA which results in a phosphodiester bond being formed and AMP being released

Energetic cost → hydrolysis of two phosphate bonds at step 1 to form one phosphodiester bond at step 3

DNA cloning

Clone → A copy or imitation of something ( eg. organism genetically identical to another, two or more identical DNA molecules)

Cloning → Ability to construct recombinant DNA molecules and maintain them in cells, generating clones

What is needed for this?

Insert DNA → A DNA fragment generated by enzymatic activity (eg. restriction enzymes) or physical treatment (eg. mechanical fragmentation of a large DNA molecule)

Vector → A DNA molecule that provides the information necessary to propagate the cloned DNA fragment, commonly a closed-circular double-stranded plasmid DNA molecule

  • The use of phosphatase can be avoided by the use of two different, non-compatible restriction enzymes

  • Ends of the vector cannot anneal as the overhang bases are not compatible

  • Ligation only occurs with the fragment

  • The fragment can only insert in one direction for the same reason

What happens when restriction sites do not occur at the right place in the fragment you want to close?

  • Any blunt end can be ligated to another blunt end, even if generated with different restriction enzymes

  • Ligation of blunt ends is less efficient than ligation of sticky ends

Oligonucleotide Linkers

  • Linkers are short duplex oligonucleotides that contain a restriction endonuclease cleavage site and can be ligated to a double stranded DNA fragment in order to create a new restriction site

Introducing DNA into bacteria to propagate and maintain cloned DNA fragments 

  • Introduction of DNA (vectors or ligation reactions) into the cell

  • Allows isolation of individual clones from complex mixtures of recombinant DNA

  • Mixed population of DNA is segregated into its individual components

Basic Methods to introduce DNA into bacteria

Chemical transformation of competent cells:

Electroporation

Protoplasts transformation

Main features of E. coli strains used in molecular cloning

  • Partially or totally deficient in native E. coli DNA restriction systems

  • Exonuclease and recombination activities reduced or abolished

  • Activity of proteases is abolished 

Introducing DNA into Mammalian cells