restriction endonucleases by Irfan Khan

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Site specific Cleavage by Restriction Endonucleases b y P.Irfan Khan [email protected]

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Restriction endonucleases RESTRICT viruses Viral genome is destroyed upon entry Restriction endonuclease = Restriction enzymes Endo (inside), nuclease (cuts nucleic acid) Restriction endonuclease recognizes a short and specific DNA sequence and cuts it from inside. The specific DNA sequence is called recognition sequence Introduction... [email protected]

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1952-53: Luria and Human discovered the phenomenon of restriction and modification Named as host-induced, or host-controlled, variation. In 1968 Matthew Meselson and Robert Yuan reported that they discovered an enzyme in E.coli K-12 –recognize and digest foreign DNA. They coined the term “ RESTRICTION ENDONUCLEASE ” to refer to this enzyme. Discovery ... [email protected]

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Bacteriophages varied in their ability to grow on different strains of E.coli . Once growth was achieved on one host strain, the phages could continue to grow happily on this strain. However, the phages were now restricted in their ability to grow on other strains. Restriction??? [email protected]

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Smith and Nathans (1973) proposed enzyme naming scheme three-letter acronym for each enzyme derived from the source organism First letter from genus Next two letters represent species Additional letter or number represent the strain or serotypes For example. the enzyme Hind II was isolated from Haemophilus influenzae serotype d. Nomenclauture ... [email protected]

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Enzyme Organism from which derived Target sequence (cut at *) 5' -->3' Bam HI Bacillus amyloliquefaciens G* G A T C C Eco RI Escherichia coli RY 13 G* A A T T C Hind III Haemophilus inflenzae Rd A* A G C T T Mbo I Moraxella bovis *G A T C Pst I Providencia stuartii C T G C A * G Sma I Serratia marcescens C C C * G G G Taq I Thermophilus aquaticus T * C G A Xma I Xanthamonas malvacearum C * C C G G G [email protected]

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Restriction-modification (R-M) system Endonuclease activity: cuts foreign DNA at the recognition site Methyltransferase activity: protects host DNA from cleavage by the restriction enzyme. Methyleate one of the bases in each strand Restriction enzyme and its cognate modification system constitute the R-M system R-M system [email protected]

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Bacteria protect their self DNA from restriction digestion by methylation of its recognition site. Methylation is adding a methyl group (CH 3 ) to DNA. Protection of Self DNA [email protected]

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Types of Cuts Blunt end Cutters Sticky-end Over-hangers [email protected]

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Blunt Ends Sticky Ends [email protected]

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Restriction enzymes are classified based on recognition sequence and methylation pattern. They are of 4 types: Type I Type II Type III Type IV Classification [email protected]

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Multi-subunit proteins Function as a single protein complex ( pentameric ). Contain two R (restriction) subunits, two M ( methylation ) subunits and one S (specificity) subunit Cofactors and activators: Mg2+ , AdoMet , ATP Cleave DNA at random length from recognition site E.g.: EcoKI , EcoAI , EcoBI , StyLTII , Type I [email protected]

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Type III Large enzymes Combination restriction-and-modification Cleave outside of their recognition sequences non- pallindromic . Require two recognition sequences in opposite orientations within the same DNA molecule. Cofactors and activators: Mg2+ , AdoMet No commercial use or availability. E.g.: EcoP151, EcoPI , HinfIII [email protected]

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Type IV Cleave only modified DNA ( methylated , hydroxymethylated and glucosyl-hydroxymethylated bases). Recognition sequences have not been well defined Cleavage takes place ~30 bp away from one of the sites. Sequence similarity suggests many such systems in other bacteria and archaea . e.g.: EcoKMcrBC [email protected]

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Type II Most useful for gene analysis and cloning More than 3500 REs Recognize 4-8 bp sequences Need Mg 2+ as cofactor Cut in close proximity of the recognition site Homodimers ATP hydrolysis is not required Exhibit great diversity. [email protected]

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Type Structure Cofactor Recognition sequence Cleavage site examples Type II Homodimer (2 R-S) Mg2+ pallindromic Defined, Within recognition site EcoRI , BamHI , HindIII , KpnI , NotI , PstI , SmaI , XhoI Type IIb Heterotrimer (2 R-M, 1S) Mg2+, Adomet Bipartite, interrupted Defined symmetric cut leaves 3’ over hangs BcgI , Bsp24I, BaeI , CjeI , and CjePI [email protected]

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Type Structure Cofactor(s) Recognition sequence Cleavage site Examples Type IIe Homodimer (2R-S), Monomer (R-S) Mg2+ pallindromic Defined Short distance away NaeI , NarI , BspMI , HpaII , Sa II, EcoRII Type IIs Monomeric (R-S0 Mg2+ Non- pallindromic Outside of Recg . site FokI , Alw26I, BbvI , BsrI , EarI , HphI , MboII , [email protected]

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The process involves: Non-specific DNA binding Target site location Recognition Catalysis Release. Mechanism of action of RE: [email protected]

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The biological function of RE is based on the fast cleavage of an invading phage DNA. Cleavage must occur before the phage DNA is methylated . This requires very rapid target site location, which is not an easy task given the large excess of non-specific over specific sites on the DNA. To overcome this problem, RE seem to have developed a common strategy for facilitated target site location: the protein very quickly binds non-specifically anywhere to the DNA and then scans the DNA in search of its recognition site. Target site location: [email protected]

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Three principally different, but not mutually exclusive, mechanisms can account for the efficiency of target site location by DNA-binding proteins: ( i ) ‘sliding’(linear or one-dimensional diffusion), (ii)‘jumping’ or ‘hopping’(three-dimensional diffusion) (iii) intersegment transfer. [email protected]

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[email protected]

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[email protected]

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The ease with which a DNA sequence can be distorted at a defined site upon interaction with RE can be used for the recognition process. The structural aspects of the recognition process: Most enzymes that produce blunt ends or sticky ends with 3 ’-overhangs approach the DNA from the minor groove. that produce sticky ends with 5 ’-overhangs contact the DNA from the major groove. Specific DNA-binding is accompanied by more or less pronounced distortions of the DNA that bring functional groups of the DNA into positions required for optimal recognition. Recognition: [email protected]

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The formation of a highly co-operative hydrogen bond network is a characteristic feature of the specific protein- DNA complex of restriction RE. This H-bond network comprises contacts to the bases (‘direct read-out’), sugar-phosphate backbone (‘indirect readout’). In addition, van der Waals interactions and hydrophobic contacts are formed to the bases of the recognition sequence. most of the specific contacts are between one subunit and one half-site of the palindromic recognition sequence, a few are directed to the other half-site. A characteristic feature of the recognition process is its high redundancy [email protected]

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SN2-type mechanism comprises three steps: the preparation of the attacking nucleophile by deprotonation : The nucleophilic attack of OH - on Phosphorous leading to formation of Pentavalent transition state . k The departure of 3’ hydroxyl leaving group H 2 O + B + R’-O5’-(PO 2 ) – -O3’-R’’ OH – + BH + + R’-O5’-(PO 2 ) – -O3’-R” OH – + R’-O5’-(PO 2 ) – -O3’-R” R’-O5’-(PO 2 OH) 2– -O3’-R” -O5’-(PO 3 ) 2– -O3’- + H 2 O R’-O5’-(PO 2 ) – -O- + HO3-R” + OH Catalysis: Mechanism of Phosphodiester cleavage: [email protected].com

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The mechanistic models for DNA cleavage by RE are based on number of metal ions involved in reaction. One-metal ion mechanism: single metal ion at the active site. stabilizes the developing negative charge during the nucleophilic attack. This mechanism is, therefore, often referred to as substrate-assisted seen in EcoRI , BglII [email protected]

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Two-metal ion mechanism: two divalent metal ions used Both required to stabilize doubly charged pentavalent transition state Seen in BamHI , BgII Three-metal ion mechanism: No crystal structure with three metal ions available Based on two metal ions in three different positions of ECoRV active site. [email protected]

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A type of immune system for individual bacterial strains, protecting them from infection by foreign DNA. Restriction enzymes can be used in processes such as… Southern Blotting Restriction Fragment Length Polymorphism • DNA finger printing • Molecular diagnostics • Cloning • cDNA library construction Applications [email protected]

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A RE functions by "scanning" the length of a DNA molecule on encountering its particular specific recognition sequence, it will bond to the DNA molecule and makes cut. • Different RE’s yield different sets of cuts, but one endonuclease will always cut a particular base sequence the same way, no matter what DNA molecule it is acting on. REare of paramount importance for rDNA work and efforts are under way to make them even more useful by expanding or changing their specificity. Conclusion … [email protected]

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