Restriction Enzymes
When were restriction enzymes applied to gene editing? Who was the first to do this?
- 1972 - Herb Boyer and Stan Cohen - made the first recombinant DNA using restriction enzymes, eventually obtained that 1st patent and formed Genetech
Give the recognition sequence of EcoRV
- 5' GATATC 3'
Describe the mechanism of the CRISPR-Cas9 system.
- CRISPR-Cas9 has two main parts: guide RNA and the Cas9 endonuclease. - bacteriophage inserts dsDNA into host cell - when foreign dsDNA comes into cell, cell looks for PAM sequence (protospacer adjacent motif) that gets cut out + directly inserted into the bacterial genome @ CRISPR loci - CRISPR loci = repeat sequences, foreign DNA gets inserted between them. (CRISPR array) - can have up to 100x repeats - physically cuts out the viral DNA and stuck it into its own genome. - when RNA transcribed from that, makes primary transcript, makes tracer/guide RNA. - tracer RNA gets incorporated into Cas9 complex - tracer RNA base pairs directly with foreign DNA - Cas9 cuts foreign DNA, disabling it via improper repair
Who were the scientists to isolate and characterize the first restriction enzyme? In what year?
- Hamilton Smith and Kent Wilcox - 1970
What does RFLP stand for? How is it applied?
- RFLP = restriction length polymorphism. - due to insertions and deletions in people, the space/ amount of DNA between restriction sites is not the same across individuals - restriction enzyme chops up DNA into slightly different lengths/ cut patterns - amplify the RFLPs and run them through a gel, bam. early forensics/ paternity testing.
How does methylation protect host DNA?
- adds steric hinderance - prevents hydrogen bonding interactions necessary to promote DNA in its bent/ distorted shape that's necessary for cleavage catalysis to occur.
Cognate and non-cognate DNA bind to the active site differently. How is their binding different?
- both cognate and non-cognate DNA form extensive hydrogen bond interactions with the restriction enzyme - hydrogen bonding to the cognate DNA is more extensive, and causes the DNA to bend up and into position for the two Mg2+ to activate H2O and cleave - the distortion of the cognate DNA generates an entropic penalty - which is satisfied by the extra hydrogen bonds the cognate DNA forms that the non-cognate cannot.
What reaction do restriction endonucleases catalyze? What are the substrates, what are the products?
- catalyze the cleave of DNA's phosphodiester backbone via hydrolysis - substrates = water and DNA's phosphodiester backbone - products = 3' OH and 5' phosphoryl group
There is a loop hole in the functional pairing of restriction endonuclease + methylase to protect host DNA from being degraded. What is the loop hole? How does the bacteria compensate for it? Is there a trade-off?
- cognate DNA cannot be methylated during replication - compensated by limiting the speed of the restriction enzyme - trade off is that the enzyme needs to act fast enough to protect bacterial DNA from viral DNA, but not too fast that it will cleave cognate bacterial DNA during replication.
What is the DNA sequence recognized by restriction enzymes called?
- cognate DNA.
What are the main differences between the two types of nucleophilic attack? Where is each type observed?
- covalent intermediate (1) maintains original stereochemistry. - analogous to chymotrypsin's mechanism - direct hydrolysis (2) inverts the original stereochemistry. - analogous to aspartyl- and metallo- proteases.
Where do restriction enzymes cleave?
- depends on their type. - type II (the type we focused on) cleave within their recognition site. - other types cut outside of the recognition site.
How is the symmetry of binding sites mirrored in the structure of restriction enzymes?
- dimers - also have two-fold rotational symmetry - this facilitates the interaction of the enzyme with the palindromic sequence (alignment of two-fold axis)
How can CRISPR-Cas9 technology be used?
- double strand break can be utilized two ways: - 1. non-homologous end joining (generates a mutation in YGI) - 2. template-directed repair/ homologous end jointing (insert YGI into the genome).
Describe the key H-bond interactions between the restriction enzymes and the DNA.
- enzyme does not h-bond to the cleavage site; h-bonds to the residues flanking the cleavage site
How do restriction enzymes selectively degrade viral DNA without degrading host DNA?
- every restriction enzyme has a specific recognition sequence/ cognate DNA - every restriction enzyme has a corresponding/matching methylase that recognizes the same gene sequence - methylase methylates the host's cognate DNA sequence - this blocks the restriction enzyme from cleaving host DNA
Why is two-fold rotational symmetry necessary in the restriction enzyme?
- for cleaving both strands of DNA.
How is the divalent cation coordinated in restriction endonucleases?
- four positions: - 1. 2x aspartates - 2. one of the oxygen atoms on a phosphoryl group - 3. water
How do divalent cations contribute to restriction endonuclease activity? Which divalent cation is most common?
- function is similar to Zn2+ in carbonic anhydrase (interacts with water to lower its pKa and generate a nucleophile) - Mg2+ is most common in restriction enzymes
What technique is used to identify the presence/ location of divalent cation binding sites in proteins? What are the limitations of this technique?
- grow a crystal of the enzyme-substrate complex in the presence of the divalent cation - problem is that, to grow a crystal, enzymes must be homogenous/ all in the same state (enzymes change states/ catalyze reactions really fast, hard to slow them down enough to be homogenous) - can also grow crystals using a mutant form of your enzyme of interest that has less activity - could substitute in a different divalent cation.
What type of stereochemistry was observed after the restriction endonuclease nucleophilic attack? What did this indicate?
- inverted - single-step mechanism
How was it determined what type of nucleophilic attack mechanism restriction endonucleases use?
- looked at the stereochemistry - stereochemistry hard to look at since the phosphoryl group has two oxygens on it (ie, substrate is not a chiral centre). - introduced sulphur on to the phosphory (phosphorthioate bond); making it a stereocenter - used labelled water (with O18) to follow stereochemistry after the nucleophilic attack.
What are the specificity requirements of restriction enzymes?
- must cut foreign DNA - must not cut host DNA
Describe the structural similarities and difference of restriction endonucleases present within the same class, and the same organism.
- overall structures are not conserved and vary quite a bit outside of the active site - the catalytic core is highly conserved.
What do you call the phosphodiester bond about to be cut by the restriction endonuclease?
- scissile bond
Describe the characteristics of the recognition sites that restriction endonucleases bind to.
- sites contain inverted repeats with two-fold rotational symmetry - sites are palindromic
Describe the relationship between restriction endonuclease specificity vs efficiency.
- specificity of a restriction enzyme takes place as the level of binding - Ex., EcoRV's recognition sequence is 6 bp long; there are 4^6 possible sequences in that space - this means that, in order to be truly specific, it needs to recognize/cleave the cognate sequence 4^6 times better than non-specific sites.
Why can CRISPR be used to target genes with intense specificity/accuracy?
- the longer you make the guide RNA, the more specific it'll be - target one part of the genome specifically
How many types of CRISPR systems are there? Which type is Cas9?
- three different CRISPR systems - CRISPR-Cas9 is a type II system
What is the general function of restriction enzymes? How do they function in bacteria?
- to recognize specific dsDNA sequences and cleave them via hydrolysis - in bacteria, restriction enzymes protect the cell from harmful foreign/viral DNA (there are 10x bacteriophages: bacteria, many have dsDNA).
How many divalent metal ion binding sites are present in restriction enzymes? In EcoRV, specifically?
- up to three sites; at least one is always occupied - EcoRV has two sites
How were restriction enzymes used in genetic engineering?
- when restriction enzyme cuts, it doesn't make perfectly neat 'blunt' ends, it makes sticky ends with an overhang - get a plasmid with a restriction site in it, and treat it with the restriction enzyme. - so, what you do is cut your YGI with the same sticky ends that the restriction enzyme makes, and then YGI gets incorporated. - treat it with a ligase to seal up the nicks, and bam. transgene.
Describe the two possible nucleophilic attack mechanisms that restriction endonucleases might use to cleave DNA's phosphodiester backbone.
-1. covalent intermediate. two-step mechanism; original stereochemistry is maintained through 2x inversions -2. direct hydrolysis. one-step mechanism; pentacoordinate transition state formed with trigonal bipyramidal geometry centered at the phosphorous atom. stereochemical inversion.
