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Department of Chemistry and Biochemistry University of Lethbridge Biochemistry 4200 II. Macromolecular Interactions Structure and Mechanism III Restriction Endonucleases Restriction Endonucleases Their principal biological function is the protection of the host genome against foreign DNA (in particular bacteriophage DNA) Restriction endonucleases occure ubiquitously among prokaryotes. They are part of the restriction-modification (RM) system, which comprises an endonuclease and a methyl transferase activity, endonuclease acts on the foreign DNA (defined recognition sequence) methyl transferase acts on the host DNA (defined recognition sequence) Almost all DNA methylases target the adenosine base. 1 Restriction Endonucleases There are a number of different sub classes restriction endonucleases. Type I : Recognize specific sequences and cut DNA at a nonspecific site > than 1,000 bp away. Type II: Recognize palindromic sequences and cut DNA within the palindrome. Type III: Recognize specific 5-7 bp sequences and cut DNA 24-27 bp down stream of the site. Type II restriction endonucleases are the most used class as they recognize and cut specific palindromic sequences in DNA → DNA technology Restriction Endonucleases There are a number of different sub classes restriction endonucleases. Type I : Recognize specific sequences and cut DNA at a nonspecific site > than 1,000 bp away. is a palindrome ? and cut DNA within Type II: RecognizeWhat palindromic sequences the palindrome. Type III: Recognize specific 5-7 bp sequences and cut DNA 24-27 bp down stream of the site. Type II restriction endonucleases are the most used class as they recognize and cut specific palindromic sequences in DNA → DNA technology 2 What is a Palindrome? A palindrome is anything that reads the same forwards and backwards: Mom Dad Tarzan raised Desi Arnaz rat. DNA Palindromes Because DNA is double stranded and the strands run antiparallel, palindromes are defined as any double stranded DNA in which reading 5’ to 3’ both are the same. • The EcoRI cutting site: 5'-GAATTC-3 3'-CTTAAG-5' • The HindIII cutting site: 5'-AAGCTT-3' 3'-TTCGAA-5' 3 Type II Restriction Endonucleases (orthodox) The main criterion for classifying a restriction endonuclease as type II: it cleaves specifically within or close to its recognition site it does not require ATP or GTP for its nucleolytic activity it is a homodimer of ~ 2 x 30 kDa molecular mass it recognizes palindormic sequences of 4-8 bp in length it requires Mg2+ for its nucleolytic activity it cleaves the bond between the 3’-OH and the 5’-phosphate Type II Restriction Endonucleases (orthodox) These enzymes can generate three different classes of products: Product name Example Products Blunt ends EcoRV 5’ … GAT ATC … 3’ 3’ … CTA TAC …5’ 3’ Sticky ends EcoRI 5’ … G AATTC … 3’ 3’ … CTTAA G … 5’ 5’ Sticky ends BglI 5’ … GCC NNNN NGGC … 3’ 3’ … CGG N NNNNCCG … 5’ 4 Type II Restriction Endonucleases Many type II restriction endonucleases do not conform to these narrow definitions → subdivisions are necessary Type II Restriction Endonucleases Structural similarity of the type II restriction endonucleases suggest a common (although distant) ancestor. The restriction endonuclease superfamily can be devided in two branches: The EcoRI Family bind DNA from the major groove produce sticky and with 5’-overhangs common core elements 5 Type II Restriction Endonucleases Structural similarity of the type II restriction endonucleases suggest a common (although distant) ancestor. The restriction endonuclease superfamily can be devided in two branches: The EcoRV Family bind DNA from the minor groove produce blunt ends common core elements Type II Restriction Endonucleases Within the comon core only four β-strand are absolutely conserved. Two of the strands (β2 and β3 in EcoRI and βd and βe in EcoRV) contain the amino acids residues directly involved in catalysis. common core elements 6 Interaction with DNA Restriction endonucleases interact with DNA in a complex manner A minimal scheme for the nuclease reactions involve at least five steps 1. non-specific binding to DNA 2. linear diffusion along DNA -sliding along major groove -hopping helical turns 3. specific binding 4. catalysis 5. release of products Interaction with DNA Consequently, restriction endonucleases have at least three major conformations: apoenzyme (not bound to DNA) non-specific (non-cognate) DNA binding specific (cognate) DNA binding EcoRV apoenzyme non-specific specific BamHI 7 DNA Binding and Target Site Location Restriction endonucleases bind DNA not only specifically but also, with considerably weaker affinity, non specifically. Water molecules (70-80) are released upon non-specific complex formation. Upon non-specific interaction the structure of the enzyme changes: EcoRV → allows interaction with DNA → catalytic center far away from DNA BamHI → no basespecific contacts apoenzyme non-specific complex BamHI DNA differs only in one base pair from the recognition sequence! → but non-specific binding PDBid 1ESG 8 DNA Binding and Target Site Location Non-specific DNA binding is the prerequisite for one-dimensional diffusion of the protein along the DNA. One-dimensional diffusion: Translation of the protein in a non free state along the DNA track. → sliding (helical movement along the a groove of the DNA) → hopping ( movement parallel to the DNA) Protein does not leave the DNA → intersegment transfer (requires two binding sites on the protein) DNA Binding and Target Site Location Sliding is the most important process in target site location. → Leaving the target side might also involve sliding etc. Sliding accelerates target site location: → under optimum conditions it allows for scanning of ~106 bases per binding event. Really ? 9 DNA Binding and Target Site Location Sliding is the most important process in target site location. → Leaving the target side might also involve sliding etc. Sliding accelerates target site location: → under optimum conditions it allows for scanning of ~106 bases per binding event. → but it’s a random walk →the effective sliding distance is much shorter ~ 1000 bp DNA Binding and Target Site Location Sliding is the most important process in target site location. → Leaving the target side might also involve sliding etc. Sliding accelerates target site location: → under optimum conditions it allows for scanning of ~106 bases per binding event. → but it’s a random walk →the effective sliding distance is much shorter ~ 1000 bp → ionic conditions, in particular Mg2+ influence sliding distance 10 DNA Binding and Target Site Location Sliding is the most important process in target site location. → Leaving the target side might also involve sliding etc. Sliding accelerates target site location: → under optimum conditions it allows for scanning of ~106 bases per binding event. → but it’s a random walk →the effective sliding distance is much shorter ~ 1000 bp → ionic conditions, in particular Mg2+ influence sliding distance EcoRI follows the helical pitch → does not overlook recognition sites → pauses at sites that resemble recognition sites Is it really that easy ? DNA Binding and Target Site Location Sliding is the most important process in target site location. → Leaving the target side might also involve sliding etc. Sliding accelerates target site location: → under optimum conditions it allows for scanning of ~106 bases per binding event. → but it’s a random walk →the effective sliding distance is much shorter ~ 1000 bp → ionic conditions, in particular Mg2+ influence sliding distance EcoRI follows the helical pitch → does not ovelook reckognition sites → pauses at sites that resemble recognition sites But its stopped by proteins tightly bound to the DNA or unusual DNA structures 11 DNA Recognition EcoRV system provides an excellent example for the structural changes occurring upon formation of the specific complex. apoenzyme non-specific complex specific complex All parts of EcoRV are ordered → especially the dimer interfaces and loops involved in DNA recognition DNA Recognition EcoRV apoenzyme non-specific complex specific complex Involves numerous base contacts → allows the enzyme to detect the sequence 12-20 hydrogen bonds are formed Only few water mediated contacts → increase in contact surface area between enzyme and DNA (wraps around the DNA) The B DNA conformation is distorted → here the DNA is bent by ~50° 12 DNA Recognition EcoRV apoenzyme non-specific complex specific complex Enzyme producing 3’ overhangs → mainly use β-stand and β-turn motif Enzyme producing 5’ overhangs → mainly use an α-helix and a loop Overview of Co-crystal Structures 13 Mechanism of Catalysis The catalysis of phosphodiester bond cleavage by restriction endonucleases can be considered as a phosphoryl transfer to water. Principle mechanisms of phosphoryl transfer reactions: Suggestions ? Mechanism of Catalysis Overall Reaction (EcoRV) Suggestions ? 14 Mechanism of Catalysis Both mechanisms differ in the amount of bond formation and bond breakage in the transition state. associative dissociative Alkaline phosphatase seems to achieve substantial catalysis via a dissociative transition state. Mechanism of Catalysis All type II restriction endonucleases, whose crystal structures have been determined have a catalytic sequence motif in common: … P D … D/E X K … where X is any amino acid For the great majority of typ II restriction endonucleases Mg2+ is an essential cofactor. → can be substituted with Mn2+, Fe2+, Co2+, Ni2+, Zn2+ 15 Mechanism of Catalysis The understanding of the mechanism of DNA cleavage by Type II restriction enzymes is critically dependent on the knowledge of how many Mg2+ ions are involved. … P D … D/E X K … The two carboxylates are Mg2+ ligands and the Lysine contacts the general acid (water molecule). Mechanism of Catalysis The second Mg2+ is not involved in catalysis despite the conserved contact with the phosphate backbone. Hydrolysis takes place in a single step. The reaction proceeds with an inversion of the stereochemical configuration 16 Mechanism of Catalysis The nucleophile is a OH- the general base is the substrate itself. → substrate assisted catalysis The 3’ phosphate is the general base, activating a water molecule to attack the 5’ phosphate. Mechanism of Catalysis The general acid is also a water molecule, liganded to both Mg2+ and the catalytic Lysine. The Mg2+ ion interacting with the general acid also functions to stabilize the transition state. 17 Comparison of Active Sites 18