topoisomerases

Question 1

what do topoisomerases do?

Answer 1

alter the topological state of DNA in cells

Question 2

what is decatenation?

Answer 2

where an enzyme makes a double stranded break and passes the other molecule through

Question 3

what can both DNA gyrase and topoisomerase IV do?

Answer 3

relax positive supercoils

Question 4

what can only DNA gyrase do?

Answer 4

put in positive supercoils

Question 5

what is the difference between the location of DNA gyrase and topoisomerase IV?

Answer 5

DNA gyrase does most of its work in front of the replication fork. topoisomerase IV does most of its work behind the replication fork

Question 6

what is the difference between type I topos and type II topos?

Answer 6

type I - cleaves only one strand

type II - cleaves both strands of DNA

Question 7

what is true about all topoisomerases?

Answer 7

the enzyme is linked to the DNA by formation of a phosphodiester bond between an active site tyrosine and a phosphate group on the DNA backbone

Question 8

what is important about tyrosine in the active site of topoisomerases?

Answer 8

the hydroxyl group on this residue is important for the breakage of DNA and formation of a covalent link

Question 9

what are the structural properties of E.coli DNA gyrase?

Answer 9

a heterotetramer A2B2 complex. the A protein is responsible for the breakage and reunion of DNA whilst the B protein is responsible for ATPase activity

Question 10

where does DNA sit in DNA gyrase?

Answer 10

in the ‘saddle region’, which contains positively charged pockets

Question 11

outline Gyrase-mediated DNA cleavage

Answer 11

• tyrosine forms a covalent link at the 5’ end, leaving a free 3’ OH
• this happens a second time, to form a gate where a second strand of DNA can be passed

Question 12

briefly outline the mechanism for DNA cleavage

Answer 12

1-magnesiums polarise the hydroxyl to facilitate proton uptake by histidine.
2-the oxygen is then able to perform nucleophilic attack on the phosphate of the DNA backbone.
3-an unidentified acid protonates the 3’ end of the DNA.
4-this results in the attachment of 5’ phosphate covalently to the gyrase

Question 13

what is the structural difference between DNA gyrase and other topoisomerases?

Answer 13

DNA gyrase has an additional CTD, a disk formed of six blades, each blade has four beta strands. 4 of the 6 blades have a positively charged surface

Question 14

what is fluorescence resonance energy transfer?

Answer 14

when template DNA has fluorescent molecules attached to either end. the donor is activated at a specific wavelength of light, causing it to emit light at a different wavelength, activating the acceptor molecule

Question 15

why can top IV wrap DNA but not put it in supercoils?

Answer 15

all ParC proteins lack a 7 aa sequence (QRRGGKG), which is blade 1 of the CTD. this sequence is highly conserved in gyrA

Question 16

what is the significance of the gyrA CTD?

Answer 16

if the DNAA has to wrap around the CTD, it is highly likely that the strand that gets trapped in the top hole is trapped in the same molecule

Question 17

what is the ATP usage mechanism in topoisomerases?

Answer 17

magnesium and positively charged amino acids interact with phosphate on the ATP. each phosphate group on ATP carries a heavy negative charge. the action of the water molecule cleaves off the final phosphate

Question 18

what class of topo does not use ATP?

Answer 18

type I

Question 19

what is unusual about novobiocin?

Answer 19

it is competitive inhibitor of ATP, but does not look structurally similar. it binds at an overlapping site to ATP, sterically hindering it