Flashcards in DNA Ligase and Restriction Enzymes (S2 Alvey) Deck (12):
How do we study genes?
1. Isolate a single gene
2. Amplify it
3. Modify it
4. Analyse expression
Problem: How do we do this in a practical setting?
-Borrow enzymes from nature, sometimes modifying it along the way
Linear piece of DNA can join into circular DNA using cos sites.
Cos sites are single stranded and complementary to each other. Cos stands for cohesive and they are called also sticky ends.
Genome Is circularised and it can be integrated into the host genome by the attachment site.
Att site: Attachment site on both the phage and E. coli genome, which allows recombination/integration of the phage genome into the E. coli genome.
DNA ligase is used in the process of DNA repair of the sugar phosphate backbone.
Requires the formation of covalent bonds
Most commonly used DNA ligase comes from bacteriophage T4.
-Called T4 DNA ligase.
DNA ligase action
Sticky ends are small single stranded pieces of DNA at the end of a double stranded molecule. They have unpaired hydrogen bonds and they find another complimentary sticky end and join together by a covalent bond.
-DNA ligase catalyses the joining of a 3' hydroxyl group to a 5' phosphate group to for a 5' to 3' phosphodiester bond.
DNA ligase joins DNA ends by:
-Adding an adenylate group to a lysine residue
-Transferring the adenylate to the terminal 5' phosphate group
-Phosphodiester bond formation
DNA ligase mechanism
The binding of the base pair using hydrogen bonding. This keeps the two molecules in close contact for the reaction to occur.
The DNA ligase covalently binds via a lysine to AMP/an adenylate group.
The DNA ligase donates the AMP to the 5' phosphate group. The AMP is then bound to the phosphate group.
The 3' hydroxyl attacks, allowing release of the AMP and the phosphodiester bond is formed.
Enzyme recycling needs to occur because DNA ligase requires ATP to become active again.
DNA ligases require ATP as a cofactor
Luria and Arber
Phages grown in one bacterial host failed to grow in a different bacterial host; they were restricted.
Rare progeny phages were able to grow in the new host: they have been modified.
Restriction is due to a nuclease that degrades foreign DNA: mechanism to protect against viral infection.
λ C DNA is destroyed by K-12 host enzymes
λ C injects linear DNA into the host cell, but this is not methylated (the AT sites are naked), unlike the host DNA. The restriction endonuclease does not attack the host genome, but does attack the phage DNA. The phage DNA is cleaved meaning there is no phage progeny and the host survives. The λ has been restricted.
Methylation of host (bacterial) DNA at sites otherwise sensitive to attack by a restriction endonuclease.
Methylation pattern is maintained during DNA replication.
-If methylation pattern is the same, viruses can escape digestion by host and integrate its DNA.
λ K DNA is not destroyed by K-12 host enzymes
Adenosine residues are methylated in both the E. coli genome, and the phage genome, so restriction endonuclease cannot recognise the viral DNA to destroy it because all of the recognition sites are methylated.
-They are methylated by DNA methylase.
As the methylated DNA is not cleaved the λ K progeny is produced.
Restriction enzymes recognise short sequences of DNA and cleave it.
There are three classes of restriction enzymes; Type I, II and III
-Types I and III cleave DNA at sites away from recognition sequence
-Type II cleave both DNA strands at specific recognition sites.
Restriction endonucleases action
They bind loosely to DNA backbone (loose interaction between RE and backbone).
It slides along the strand randomly in both directions until it finds the sequence of its recognition site.
If it hasn't found the correct sequence within about 50bp, it begins 'hopping and jumping'.
The enzyme diffuses off and on the DNA, that is nearby. This doesn't mean it jumps to the next bp's in the sequence, as the DNA is curled up into a knotted structure normally, it isn't perfectly linear.
-It will diffuse onto a nearby piece of DNA, but that does not mean it will be nearby in the sequence.
It will hop and jump until it finds its correct recognition site and will bind tightly to the sequence.
It will then catalyse the digestion of the DNA into two fragments.