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Topoisomerase II
Description of the structure and function of the Topoisomerase II
| Question | Answer |
|---|---|
| What function do DNA Topoisomerases perform? | Resolve topological problems in DNA resulting from the many processes involving DNA |
| What is the difference between type I and type II topoisomerases? | Type I topoisomerases only cleave one strand of DNA whereas type II topoisomerases cleave both strands |
| What is the generalized mechanism of type II topoisomerases? | Create a break in the G-DNA, pass T-DNA through the G-DNA break, religate G-DNA |
| How does the enzyme cleave the G-DNA? What kind of intermediate is formed? | A pair of catalytic tyrosines perform nucleophilic attack on phosphodiester backbone, resulting in a covalent intermediate |
| Is the presented structure complete? | No, the structure does not model amino acids 1-407. |
| How many subunits make up the enzme? | 2; topoisomerase II is a homodimer. Each subunit has its own catalytic tyrosine |
| Which domains contact the G-DNA? | The TOPRIM (topoisomerase/primase) domain and winged helix domains |
| Where is the C-gate located? | Between the two subunits |
| What interactions does the enzyme make with its natural substrate at the catalytic site? | The side chain of Tyr782 cleaves the DNA, forming a covalent link to the 5' end of the new DNA fragment |
| Why is the G-DNA only nicked on one strand? | The G-DNA is a suicide substrate, containing a sulfur instead of an oxygen in the bridging phosphate at the active site of the G-DNA |
| What is the difference between the 2 structures solved in this paper? | The first structure does not contain the metal ions required for enzyme activity, the second one does |
| How were the two different structures obtained? | The first structure was solved at a pH of 4.5 The second structure was solved at a pH of 6.5 in the presence of Zn ions and glutaraldehyde |
| What is the role of glutaraldehyde? | To crosslink proteins to DNA |
| What is surprising about the metal ions bound to topoisomerase II? | Only metal A is involved in transition-state stabilization, metal B is not located near the active site |
| What elements of the structure form interactions with metal A? | -A non-bridging oxygen of the phosphotyrosine -the 3' ribose hydroxyl oxygen, which has been replaced by a sulfur in the suicide substrate |
| What contacts does metal B make? | A non-bonding oxygen of the -1/-2 phosphate |
| What is significant about the environment of the metals? | It is relatively hydrophobic, which will increase the strength of the hydrogen bonds formed. |
| Why does topoisomerase II only use one metal in the active site rather than 2 like most DNA-binding enzymes? | -The attacking Tyr residue has a much lower pKa than the 3' OH of nucleotide sugars. This makes the Tyr a better nucleophile. -The presence of Arg781 mimics the presence of metal B in other enzymes, stabilizing the transition state. |
| What is the first step in the catalytic mechanism? | Tyrosine 782 is deprotonated by a general base, forming a nucleophilic tyrosinate. |
| What happens after Tyr782 is deprotonated? | The tyrosinate attacks the phosphate on the G-segment backbone, forming a covalent intermediate |
| What happens after Tyr782 attacks the phosphate backbone of the G-DNA? | The exposed 3' oxyanion accepts a proton from a general acid in solution |
| How are the G- and C-gates coupled? | Upon G-DNA cleavage, there is a shift of about 6 angstroms in the vicinity of Tyr782. This movement pulls on the lever arm, closing the C-gate |
Created by:
mbell133
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