Introduction to Restriction Endonuclease
- 1. Molecular Tools in Recombinant DNA Technology: Restriction Endonucleases Namrata Singh, PhD
- 2. Restriction Endonucleases • Restriction Endonucleases are the enzymes that modify nucleic acids that provides the foundation for many molecular biology techniques. These enzymes are used to synthesize, degrade, join or remove portions of nucleic acids in a controlled and defined manner. • Many recombinant DNA technologies, which the field of biotechnology heavily relies on, are unlikely to have been developed without the discovery of these restriction enzymes. • Restriction endonucleases derive their name from a host-controlled phenomenon of restriction and modification. R-M systems comprise pairs of opposing intracellular enzyme activities: a site-specific endodeoxyribonuclease (ENase), and a DNA-methyltransferase (MTase) • Most of the restriction endonucleases recognize palindromic or partially palindromic (dyad symmetry around an axis) sites referred to as recognition sequence or recognition site. Different bacterial species make restriction enzymes that recognize different nucleotide sequences. • In some R-M systems, the restriction and the modification enzyme(s) are separate proteins that act independent of each other. While in others, the two activities occur as separate subunits, or as separate domains, of a larger, combined, restriction-and-modification enzyme.
- 3. Nomenclature of restriction endonucleases A systematic method of nomenclature has been developed for all restriction enzymes based upon the name of the organism from which they were isolated. The name given to each new enzyme conveys both the genus and the species of the bacterium from which it was isolated, the strain number, and the order in series in which the enzyme was found. For example, EcoRI is derived from Escherichia coli hence Eco. The R indicates the restriction system was originally isolated on a R-factor (a plasmid carrying an antibiotic resistance) and the Roman numeral I indicates this was the first system isolated from the strain. Derivation of the EcoRI name Abbreviation Indicates Description E Escherichia genus co coli species R RY13 strain I First identified order of their identification
- 4. BamHI BamHI was the first restriction enzyme discovered in bacterium Bacillus amyloliquifaciens, strain H while the HaeIII was the third enzyme found in Haemophilus aegyptius. Molecular structure of endonuclease BamHI
- 5. Types of restriction endonucleases Restriction enzymes are classified into four different types on the basis of subunit composition, cleavage position, sequence specificity, and cofactor requirements. However, amino acid sequencing revealed that at the 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 (StySJI M protein)
- 6. 2. Type II (REases) • Type II systems are most commom and recognize specific and symmetric DNA sequences and cleave (hydrolyzes specific 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
- 7. 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 their recognition 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 inversely oriented. • They require the presence of two inversely oriented unmethylated recognition sites for restriction to occur and they methylate 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 Mrr systems 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
- 8. 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
- 9. 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 to as 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 a DNA 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 recognition sequences. Type II enzymes cleave DNA in different ways and produce cohesive (complemenary) termini that can 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
- 10. Mechanism of cleavage Cleavage of DNA by restriction endonuclease BamHI generating 5’-staggered end Cleavage of DNA by restriction endonuclease KpnI generating 3’-staggered end Cleavage of DNA by restriction endonuclease SmaI generating blunt end
- 11. Comparison Property Type I Type II Type III Restriction and Modification Single multifunctional enzyme with R(endonuclease), M(methylase), and S(specificity) subunits Separate endonuclease (homodimer) and methylase (monomer) Separate enzymes sharing common subunit. M separately functions as methylase and with R it functions as methylase- endonuclease. Nuclease subunit structure Heterotrimer Homodimer Heterodimer Cofactors ATP, Mg2+, SAM (for cleavage and methylation) Mg2+, SAM (for methylation only) Mg2+, ATP (for cleavage), SAM (needed for methylation and stimulates cleavage) DNA cleavage requirements Two recognition sites at any orientation Single recognition site (Palindrome) Two recognition sites (head to head orientation) Recognition site Bipartite and asymmetrical Short sequence (4-8bps), often palindromic Asymmetrical sequence (5-7bps) Site of methylation At recognition site At recognition site At recognition site DNA translocation Yes No No
- 12. 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 a defined DNA fragment could be moved from one organism to another making it very useful in traditional 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 and parental testing which is very important for diagnosing DNA sequence content and are used in fields as 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