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Structural Molecular Biology > Roper - Topoisomerases > Flashcards

Roper - Topoisomerases Flashcards

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1
Q

What is the difference between topoisomerases 1 and 2?

A

Topoisomerase 1 enzymes cut one strand

Topoisomerase 2 cuts both strands

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2
Q

What drugs target topoisomerases?

A
  • 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
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3
Q

What do topoisomerases do on a molecular level?

A
  • 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.
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4
Q

What do topoisomerases do on a general level?

A
  1. Unwind DNA/relax supercoiling.
  2. Decatenate DNA (or separate them when two or more circles of DNA are linked)
  3. Take out knots although knots don’t really occur in nature.
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5
Q

What are the two mechanisms used by topoisomerases and which enzyme uses which?

A

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.

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6
Q

What does Adduct mean?

A

the product of a direct addition of two or more distinct molecules, resulting in a single reaction product containing all atoms of all components

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7
Q

What adducts are produced from topoisomerases?

A

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.

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8
Q

What do Type 1 Topoisomerases do?

A

These break one strand of DNA and swivel it around the other.

Type 1A topoisomerases form 5’ adducts.
Type 1B form 3’ adducts.

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9
Q

What structural features are important to the function of Type 1 Topoisomerases?

A

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.

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10
Q

Draw the type1 mechanism

A

POSTER - Draw the type1 mechanism

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11
Q

Describe the type 1 topoisomerase mechanism

A

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.

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12
Q

How has structural biology revealed the mechanism of type 1 topoisomerases?

A
  • 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.
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13
Q

What do Type 2 Topoisomerases do?

A 
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
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14
Q

What are prokaryotic type 2 topoisomerases called?

A

DNA gyrases

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15
Q

What are DNA gyrases?

A

prokaryotic type 2 topoisomerases

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16
Q

What do DNA gyrases do?

A

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.

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17
Q

How do type 2 topoisomerases reconnect double strands?

A

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.

18
Q

How do you lock topoisomerases into an ATP bound state?

A

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.

19
Q

What is the mechanism by which type 2 topoisomerases work?

A

They seem to use a helix turn helix motif in the active site region which reaches into the DNA.

20
Q

What structural features are important to the function of Type 2 Topoisomerases?

A

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).

21
Q

What is the mechanism by which topoisomerase 2 enzymes work?

A

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.

22
Q

What study revealed a rigid bodied movement between the ATP bound and unbound state in the ATPase domain in topoisomerases?

A

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.

23
Q

What is the role of Lysine in topoisomerases?

A

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.

24
Q

What is the main conclusion that can be drawn about topoisomerases from structural studies?

A

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.

25
Q

Which study proposed the two-gate model of a type II topoisomerase mechanism?

A

James Berger et al (2007) found the structural basis for gate-DNA recognition and bending by type IIA topoisomerases in yeast by looking at the structure of the TOPRIM, WHD and Tower alone.

26
Q

What is the gene for type IIA topoisomerase like?

A

It has the ATPase domain on the N terminus.
Then has TOPRIM, WHD and ‘Tower’ domains.
Then there is the C terminal domain (CTD).

27
Q

What is TOPRIM

A

A conserved Rossman fold domain found in topoisomerase and DNA primases – structural motif common in proteins which bind and manipulate DNA

28
Q

What is WHD?

A

winged helix domain containing active site tyrosine

29
Q

What is ‘Tower’?

A

an alpha/beta domain adjacent to the WHD domain
it is composed of a number of different positively charged lysine residues. These interact with the negatively charged DNA backbone and stabilise it in this bent conformation.

30
Q

Topoisomerases bend DNA. How do they do this?

A

In a beta hairpin loop.
Two beta sheets in antiparallel conformation (with lots of H bonds between them) with a loop that has a hydrophobic isoleucine in it from the TopoII that sticks into the groove of DNA to distort the DNA structure. The active site tyrosine is located at the bend.
TOWER and the beta hairpin loop stabilise the bent DNA and channels it towards the active site tyrosine.

31
Q

How many active site tyrosines are there in TopoII?

A

two, one from each monomer in the dimer.

they come close together when DNA binds

32
Q

Which study tells us how ATP hydrolysis is linked to the translocation of DNA?

A

James Berger’s paper from 2012, which looked at the structure of ATPase domain with TOPRIM, WHD and TOWER. The protein was locked in an ATP and DNA bound state and the structure was found to 4.4A (not the best so pinch of salt).

33
Q

How do you lock topoisomerases into a DNA bound state?

A

Replace phosphodiester bond with a phosphorothioate site. This is when phosphorus is replaced with sulphur which creates a stable linkage with the protein that binds to it.

34
Q

What were the key observations from James Berger’s 2012 study?

A
  • The ATP domain sits on top of the DNA cleavage domain
  • The two holes are off set by around 45 degrees
  • The polypeptide for one protomer wraps around the other (domain swapping)
35
Q

What is domain swapping?

A

When the ATPase region of one of the monomers forms a linkage with the TOPRIM fold of the other monomer. So the ATPase domain in one is connected to the DNA binding domain in the other.

36
Q

What proteins have domain swapping?

A

Topoisomerases and Hsp90.

Hsp90 has an intertwined dimer structure with an ATPase domain on top of a much larger DNA binding domain.

37
Q

What is a K loop?

A

A loop containing a lot of lysines (3 or 4 in a row)

It creates an electrostatic charge.

38
Q

How was the K loop found to be functionally significant?

A

Using gel-based assays which assessed the relaxation of supercoiled DNA.
Tests were done with wildtype and mutant enzymes with K converted to alanine
Test 1: fixed amount of DNA with increasing protein
Test 2: assessed relaxation over time.
Wildtype enzyme found to relax supercoiled DNA quickly.
Mutant had removed functionality.

39
Q

What role does the K loop play in topoisomerases?

A

K loop enhances ATPase activity/ATP hydrolysis

40
Q

What affects topoisomerase activity?

A

Different reaction rates occur with different concentrations of ATP (as ATP increases, reaction rate is faster)
But DNA binding stimulates ATPase activity - there is no activity without DNA.
Amount of DNA does not affect ATPase activity.
DNA binding affects ATP hydrolysis and this effect is mediated by residues in the K loop region.

41
Q

How are ATP molecules used in the revised two gate model?

A

One ATP molecule is hydrolysed to cut the G segment and move the T segment across.
The other is used to open the C terminus and let the T segment out.
The hydrolysis of ATP in one protomer causes a conformational change in the other segment.

42
Q

What is the revised two gate model?

A
  1. G segment DNA binds
  2. 2 ATP molecules bind which promotes T segment binding
  3. The first ATP hydrolyses while G segment is cleaved and T segment passes. ATP domains swivel to prevent reverse recation
  4. G segment is religated. Second ATP is hydrolysed with help of K loop, which triggers conformational change to release T segment and reset enzyme.

Structural Molecular Biology (5 decks)

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