Molecular Tools in Recombinant DNA Technology: Restriction Endonucleases

Namrata Singh, PhD

Restriction Endonucleases

• Restriction Endonucleases are the enzymes that modify nucleic acids that provides the foundation for many molecularbiology techniques. These enzymes are used to synthesize, degrade, join or remove portions of nucleic acids in acontrolled and defined manner.

• Many recombinant DNA technologies, which the field of biotechnology heavily relies on, are unlikely to have beendeveloped without the discovery of these restriction enzymes.

• Restriction endonucleases derive their name from a host-controlled phenomenon of restriction and modification. R-Msystems comprise pairs of opposing intracellular enzyme activities: a site-specific endodeoxyribonuclease (ENase), and aDNA-methyltransferase (MTase)

• Most of the restriction endonucleases recognize palindromic or partially palindromic (dyad symmetry around an axis) sitesreferred to as recognition sequence or recognition site. Different bacterial species make restriction enzymes that recognizedifferent nucleotide sequences.

• In some R-M systems, the restriction and the modification enzyme(s) are separate proteins that act independent of eachother. While in others, the two activities occur as separate subunits, or as separate domains, of a larger, combined,restriction-and-modification enzyme.

Nomenclature of restriction endonucleases

A systematic method of nomenclature has been developedfor all restriction enzymes based upon the name of theorganism from which they were isolated. The name given toeach new enzyme conveys both the genus and the speciesof the bacterium from which it was isolated, the strainnumber, and the order in series in which the enzyme wasfound.

For example, EcoRI is derived from Escherichia coli henceEco. The R indicates the restriction system was originallyisolated on a R-factor (a plasmid carrying an antibioticresistance) and the Roman numeral I indicates this was thefirst system isolated from the strain.

Derivation of the EcoRI name

Abbreviation Indicates Description

E Escherichia genus

co coli species

R RY13 strain

I First identifiedorder of their identification

BamHI

BamHI was the first restriction enzymediscovered in bacterium Bacillusamyloliquifaciens, strain H while the HaeIIIwas the third enzyme found in Haemophilusaegyptius.

Molecular structure of endonuclease BamHI

Types of restriction endonucleases

Restriction enzymes are classified into four different types on the basis of subunit composition, cleavageposition, sequence specificity, and cofactor requirements. However, amino acid sequencing revealed that atthe molecular level there are many more than four different types.

1. Type I

• Type I R-M systems have been found in E.coli, Citrobacter, and Salmonella

• These are multisubunit enzymes that function as a single protein complex

• Grouped in three major families

• Have little practical value since they don’t produce discrete restriction fragments or distinct gel-banding patterns

Molecular structure of Type I Restriction endonuclease (StySJIM protein)

2. Type II (REases)

• Type II systems are most commom and recognize specific and symmetric DNA sequences and cleave (hydrolyzesspecific phosphodiester bonds) at constant positions in the presence of Mg2+ at or close proximity to the recognition

sequence.• They may act as monomers, dimmers, or tetramers, and are independent of methyl tranferases• Type II enzymes differ in amino acid sequence from one another, and indeed from other known proteins• Mostly type II enzymes recognize DNA sequences that are symmetric because they bind to DNA as homodimers but

few binds to heterdimers too• These are further divided into Type IIA, IIB, IIC, IIE, IIF, IIG, IIH, IIM, IIP, IIS and IIT

Endonuclease BgII Endonuclease NaeI endonuclease FokI Endonuclease EcoRI

* All pictures courtsey wikipedia commoms in this presentation

3. Type III

• Type III systems are the large combination R-M enzymes

• They recognize short, non-palindromic sequences (methylated on one strand), and cleave outside of theirrecognition sequences and require AdoMet and ATP cofactors for their roles in DNA methylation and restriction,respectively.

• Type III restriction enzymes (e.g., EcoP15) recognize two separate non-palindromic sequences that are inverselyoriented.

• They require the presence of two inversely oriented unmethylated recognition sites for restriction to occur and theymethylate only one strand of DNA

• They cleave DNA in the immediate vicinity of their recognition sites, e.g., EcoP1, EcoP15, HinfllI

4. Type IV

• Type IV enzymes recognize modified, typically methylated DNA and are exemplified by the McrBC and Mrrsystems of E. coli

5. Type V

• Type V enzymes can cut DNA of variable length and utilize guide RNAs to target specific non-palindromic

sequences found on invading organisms. This characteristics makes them promising tool for genetic engineering

Artificial restriction enzymes

• Artificial restriction enzymes can be generated by fusing a natural or engineered DNA domain to nuclease domain

• These enzymes can be modified to bind desired DNA

• Zinc fingers and TAL (transcription activator-like) effectors are examples of artificial restriction enzymes

• There has been an ongoing development of artificial ribonucleases to cleave RNA

Repair outcomes of a genomic double-strand break for ZFN cleavage

Mechanism of action

• The actions of restriction endonucleases vary. However, in general, the process involves recognition of binding site,binding of enzyme dimer to the DNA, cleavage of DNA, and finally release of enzyme

• Restriction endonucleases must show tremendous specificity at two levels

o First, they must cleave only DNA molecules that contain recognition sites (hereafter referred toas cognate DNA) without cleaving DNA molecules that lack these sites

o Second, restriction enzymes must not degrade the host DNA

• R. J Roberts coined the term Isoschizomer (same cutter) for enzymes recognizing and cleaving DNA at specific sites.While, those enzymes cleaving DNA at different sites within identical nucleotide sequences are called Neoschizomers

• Type I restriction–modification systems are multifunctional complexes. These heteromeric complexs can act as aDNA methyltransferase, a DNA-dependent ATPase, a DNA translocase and a restriction endonuclease

• Type II restriction endonucleases cleave double-stranded DNA at specific sites within or close to their recognitionsequences. Type II enzymes cleave DNA in different ways and produce cohesive (complemenary) termini thatcan be used to create novel DNA molecules. Three types of termini can be generated.

o 5' staggered ends,

o 3' staggered ends, and

o Blunt ends

• DNA cleavage by type III restriction endonucleases requires two inversely oriented asymmetric recognition sequences and results from ATP-dependent DNA translocation and collision of two enzyme molecules

Mechanism of cleavage

Cleavage of DNA by restriction endonuclease BamHIgenerating 5’-staggered end

Cleavage of DNA by restriction endonuclease KpnIgenerating 3’-staggered end

Cleavage of DNA by restriction endonuclease SmaI generating blunt end

Comparison

Property

TypeI

TypeII

TypeIII

RestrictionandModification

Singlemultifunctional

enzymewithR(endonuclease),

M(methylase),andS(specificity)subunits

Separate

endonuclease(homodimer)and

methylase(monomer)

Separateenzymessharingcommon

subunit.Mseparately

functionsasmethylaseandwith

Ritfunctionsasmethylase-

endonuclease.

Nucleasesubunit

structure

Heterotrimer

Homodimer

Heterodimer

Cofactors

ATP,Mg2+,SAM(for

cleavageandmethylation)

Mg2+,SAM(for

methylationonly)

Mg2+,ATP(forcleavage),SAM

(neededformethylationand

stimulatescleavage)

DNAcleavagerequirements

Tworecognitionsitesat

anyorientation

Singlerecognitionsite(Palindrome)

Tworecognition

sites(headtoheadorientation)

Recognitionsite

Bipartiteandasymmetrical

Shortsequence(4-8bps),often

palindromic

Asymmetrical

sequence(5-7bps)

Siteofmethylation

Atrecognitionsite

Atrecognitionsite

Atrecognitionsite

DNAtranslocation

Yes

No

No

Applications

• The ability of restriction enzymes to reproducibly cut DNA at specific sequences has led to the widespread use of these tools in many molecular biology techniques.

o Cloning: In combination with DNA ligases, REases facilitate “cut and paste” workflow where adefined DNA fragment could be moved from one organism to another making it very useful intraditional cloning experiments.

o DNA Mapping: It helps in detection of single nucleotide polymorphisms and insertions/deletions,applications that include identifying genetic disorder loci, genetic diversity of populations andparental testing which is very important for diagnosing DNA sequence content and are used in fieldsas disparate as criminal forensics and basic research

o Epigenetic Modifications

o In vitro DNA Assembly Technologies: Synthetic biology creates biological systems for the study of the processes and the creation of useful biological devices. Biobrick, Golden Gate and Gibson assembly are the novel technologies that uses restriction endonucleases.

o In vivo gene editing, creating nicks in DNA, inserting foreign genes and constructing libraries