Roper - Topoisomerases Flashcards
What is the difference between topoisomerases 1 and 2?
Topoisomerase 1 enzymes cut one strand
Topoisomerase 2 cuts both strands
What drugs target topoisomerases?
- Anti-cancer drugs (such as camptothecin)
- Antibiotics (such as novobiocin and nalidixic acid) – usually a DNA gyrase/topoisomerase II poison – very good because it is difficult to develop resistance to it as it affects DNA topology
What do topoisomerases do on a molecular level?
- Cleave phosphodiester bonds only through nucleophilic attack from specific tyrosine residues
- The pka of tyrosine is about 9.5 or 10 so it is brought back down to physiological pH when ionised.
What do topoisomerases do on a general level?
- Unwind DNA/relax supercoiling.
- Decatenate DNA (or separate them when two or more circles of DNA are linked)
- Take out knots although knots don’t really occur in nature.
What are the two mechanisms used by topoisomerases and which enzyme uses which?
Swivel mechanism (used by Topo I) is only used to unwind supercoils
Strand Passage (used by Topo II) is used to unwind supercoils and decatenane and untie knots.
What does Adduct mean?
the product of a direct addition of two or more distinct molecules, resulting in a single reaction product containing all atoms of all components
What adducts are produced from topoisomerases?
They both make a 5’ adduct and a 3’ adduct.
In both cases, the enzyme via tyrosine creates a bond with the phosphate on the DNA backbone. 5’ adducts occur off the phosphate on the 5th position of the ribose ring and 3’ adducts occur off the phosphate on the 3rd position.
What do Type 1 Topoisomerases do?
These break one strand of DNA and swivel it around the other.
Type 1A topoisomerases form 5’ adducts.
Type 1B form 3’ adducts.
What structural features are important to the function of Type 1 Topoisomerases?
Most have a cleavage/strand passage domain in the N terminal region.
Followed by a metal (usually zinc but sometimes magnesium) DNA binding domain.
The C terminus contains the active site tyrosine.
The N and C termini open like a jaw to let DNA in and out and change the linking number by 1.
Draw the type1 mechanism
POSTER - Draw the type1 mechanism
Describe the type 1 topoisomerase mechanism
Protein binds with one strand of DNA between the C and N terminus.
Phosphodiester bond forms between active site tyrosine and one strand, breaking the old phosphodiester bond.
The other strand is passed through the new gap and swivels round.
How has structural biology revealed the mechanism of type 1 topoisomerases?
- Solved the structure of human topoisomerases type 1b in the presence and absence of DNA.
- Found that a big chunk of it was missing: the N terminus or the first 174 amino acids.
- Found a big hole in the middle where DNA is thought to go in
- Changed tyrosine to phenylalanine so same amino acid without hydroxyl group. Meant that the protein could bind DNA but not cleave it. Evidence for tyrosines role.
What do Type 2 Topoisomerases do?
These break two strands of DNA and pass. They are ATP dependent enzymes. Changes the linking number by 2. Eukaryotic Topo2s relax DNA only. Type 2A = make 3’ adducts Type 2B = make 5’ adducts
What are prokaryotic type 2 topoisomerases called?
DNA gyrases
What are DNA gyrases?
prokaryotic type 2 topoisomerases
What do DNA gyrases do?
Gyrases can relax and supercoil. They do it depending on ATP state. Relaxation is atp-independent whereas supercoiling is ATP dependent.
The same single polypeptide chain is broken into two and form a heterotetramer (A2B2).
Prokaryotic B subunits (GyrB) are equivalent to the N terminus of eukaryotic type 2 topoisomerases.
And Gyr A line up with the C terminus.
How do type 2 topoisomerases reconnect double strands?
Type 2 Enzymes cleave DNA on both strands with a 4bp stagger. It is like a restriction enzyme but with no specificity. Cleavage is via tyrosine. Huge conformational changes occur to move strands through gap through the centre of the protein.
How do you lock topoisomerases into an ATP bound state?
With a non-hydrolysable ATP analogue like AMP-PNP. Between the beta and gamma phosphates is a nitrogen so its Adenosine-phosphate-nitrogen-phosphate instead of three phosphates. So the bond between gamma and beta is non hydrolysable. There is also a version with carbon.
What is the mechanism by which type 2 topoisomerases work?
They seem to use a helix turn helix motif in the active site region which reaches into the DNA.
What structural features are important to the function of Type 2 Topoisomerases?
ATP binding site is in the N terminus of GyrB
When in dimer formation, there is a hole where the DNA is threaded through
Conformational changes switch it from an open and closed state.
ATP dependent N terminus is sat on top of the C terminal domain (DNA cleavage and strand passage domain).
What is the mechanism by which topoisomerase 2 enzymes work?
The two gate model:
a. The G (gate?) segment of DNA enters via the top of the protein.
b. It binds to the active site tyrosine in the enzyme core, causing a conformational change.
c. The T (transfer) segment can then bind into the top of the protein which closes behind it
e. The G segment is broken by the active site tyrosine, making a gap that the T segment can pass through.
f. The G segment is reformed, and the T segment is on the other side.
What study revealed a rigid bodied movement between the ATP bound and unbound state in the ATPase domain in topoisomerases?
Wei et al showed the nucleotide-dependent domain movement in the ATPase domain of human type IIa topoisomerase
It was expressed in e. coli, and it is equivalent to DNA gyrase.
Protein was crystallised in 2 conditions; with ADP (ATP unbound) and with AMP-PNP (represents ATP bound).
Comparing them showed a conformational change upon ATP hydrolysis. Opens up by 6 angstrom to 17. This allows for strand passage.
What is the role of Lysine in topoisomerases?
Responsible for ATP binding.
It is a highly conserved amino acid, always found in a QTK loop in the transducer domain.
Lysine residue reaches in from the transducer module to the ATP and forms a salt bridge with one of the phosphates in ATP
Mutagenesis studies moved the lysine to different positions. It was unable to form the ATP bound conformation without it.
What is the main conclusion that can be drawn about topoisomerases from structural studies?
There is a a rigid bodied movement between the ATP bound and unbound state in the ATPase domain.
ATP binding, hydrolysis and phosphate release is associated with the opening of the cavity in the N terminal region with rotates towards the middle of the protein.