Topoisomerases

Question 1

what is topology and how does it relate to DNA

Answer 1

Topology = properties of an object that remain constant, but the form may change by e.g. twisting, knotting, stretching and bending
Changes in DNA form caused by knotting, stretching and bending = Catenane – two DNA molecules joined together in a circular form. Very important to allow DNA replication to occur efficiently.

Question 2

What do topoisomerase do ?

Answer 2

Single strand = Bonds in one DNA strand must be broken to inter convert these forms
Double strand = Bonds in both DNA strands must be broken to inter-convert these forms

Question 3

What is difference between the classes of topoisomerase enzymes

Answer 3

type 1 = one a single DNA is cut
type 2 = both strands are cut

Question 4

What is the topological constraints on DNA

Answer 4

○ DNA strand separation essential for Transcription, replication
○ Double helical structure means that there is no rotation of the strands
○ During strand separation (e.g replication) supercoiling of DNA occurs
○ Without Topoisomerases, supercoiling stalls replication & transcription
○ Negative supercoiling leads to abnormal DNA structures (D loops, R loops, Z-DNA)
○ For efficient replication of the genome, topoisomerases are key

Question 5

What are human topoisomerase function

Answer 5

○ Topoisomerase I (Type I enzyme) = DNA replication and transcription - Topo I is part of transcription complexes. (k/o mouse-lethal)
○ Topoisomerases IIα and IIβ (Type II enzymes)
= α: chromosome segregation, resolves intertwined chromosomes at mitosis (mouse-lethal)
= β- neuronal/development
○ Chromatin condensation and decondensation.
○ IIβ essential for transcription initiation

Question 6

How do topoisomerase II poisons work and what are the current uses

Answer 6

• reaction is poisoned not inhibited
• catalytic inhibitors did exist but there was no reaction but no DNA damage
• Uses show target for cancer cells and design drugs to inhibit these targets

Question 7

What is etoposide

Answer 7

Top II poison, weak anti-tumour activity
- created etoposide then teniposide

Question 8

what are the functions of topoisomerase II poisons

Answer 8

• Topoisomerase II is a two homodimers. Topoisomerase breaks the two strands of DNA and is covalently bound to the DNA (phosphodiester bond). Enzyme changes conformation to allow second DNA strand to pass through. DNA then religated and topo II leaves
• Etoposide forms a cleavable complex (phosphotyrosyl) with the topo II and DNA. This blocks the reaction and stops the Topo II from opening and closing to let the second strand through.

Question 9

how are cleavable complexes formed in the cell detected

Answer 9

Alkaline elution (Trapped in agarose DNA immunostained TARDIS)
- Live cells embedded in layer of agarosegel
- Cells lysed to remove proteins, etc, leaving just DNA
- Add fluorescent antibodies that bind to Topoiso protein
- Topoiso bound to DNA – detected by immunofluorescence
- Topoisomerase poisons cause enzyme molecules to become bound to the DNA – cleavable complexes
□ DNA – detected by fluorescent dye (blue)
□ Topoisomerase bound to DNA – antibodies detected by fluorescence (green)

Question 10

What are anthracyclines

Answer 10

• The flat 4-ring structure enters (intercalation) between bases in DNA – distorts double helix, blocks (poisons) Topo II
• Free radical formation hydroxy radicals formed -damage DNA and cell membranes- cardiotoxicity
  □ Doxorubicin - widely used for solid tumours e.g. breast, lung, bladder
  □ Daunorubicin, Idarubicin - leukaemia

Question 11

Why cant patients be given camptothecin

Answer 11

Though found to kill tumours cells by poisoning topoisomer I poisoning
it was proven to be to toxic- phase I/II trails (severe, unpredictable toxicities)
Derivative = overcome toxicities

Question 12

what is Irinotecan

Answer 12

Topoisomerase I poison
a pro-drug which forms SN-38 (active form) by carboxylesterases CES1/CES2

Question 13

how is SN-38 metabolised

Answer 13

primarily in the liver to the inactive SN-38 glucuronide by uridine diphosphate glucuronosyltransferase (UGT1A1)
excreted in bile

Question 14

why is there a large variability in irinotecan response in patients

Answer 14

polymorphisms - can cause severe toxicity (neutropenia)
- Look at AUC of irinotecan, SN38 and SN38G
- These data prompted FDA to alter drug labelling of CPT-11 (now includes homozygosity for the UGT1A128 as a risk factor for severe neutropenia)
30% of the variability in SN-38 AUC is explained by ABCC1 1684TC, ABCB1 IVS9 –44AG, and UGT1A193

Question 15

what is the function of irinotecan and what is the mechanisms

Answer 15

Breaks single strand of DNA, forms covalent bonds
Allows rotation of one strand around another
Camptothecin forms a cleavable complex
-> Leads to downstream effects

Question 16

How does topoisomerase enzyme cause cytotoxicity

Answer 16

○ Results not simply from inhibition of enzyme activity BUT From the formation of stabilised cleavable complexes
○ Topoisomerase poisons convert normally transient enzyme intermediates into permanent DNA damage

Question 17

What happens to cells after topoisomerase

Answer 17

DNA damage -> Delay of cell cycle progression (repair/removal of damage)
either
- cell division not possible (mitotic catastrophe)
- cell survives
- Apoptosis triggered by damage

Question 18

T/F topoisomerase poisons are specific

Answer 18

False
non-specific hence sever toxic side effects

Question 19

Why do cytotoxic drugs preferentially attack tumours

Answer 19

• Cells are more sensitive during DNA replication - -
• Many dividing cells in tumours (also in normal tissues)
• Tumour cells often defective in check-point control + DNA repair

Question 20

What are the resistance mechanism involved in topoisomerase poison treatment

Answer 20

• less access to target (less drug in cell)
• Altered target or less target
• altered response to DNA damage

Question 21

How does less access to target causes resistance form topoisomerase poisons

Answer 21

Over expression of drug export pumps - Muti drug Resistance MDR1
□ P-glycoprotein (Membrane export pump protein overexpressed) Acts on large hydrophobic molecules, these enter cell by passive diffusion, many drugs, including etoposide

Question 22

How does altered/less target causes resistance form topoisomerase poisons

Answer 22

□ Reduced level of Topo I/II enzyme
OR
□ Altered enzyme – not affected by drug (mutation?)
□ Less DNA damage formed (i.e. less cleavable complex formation)
□ Confers resistance to many different drugs that target Topo II
□ Classical multidrug resistance (overexpression of drug export pumps

Question 23

How does an altered response to DNA damage cause resistance to topoisomerase poisons

Answer 23

• Enzymes that remove covalently-bound topoisomerase.
  □ TDP1 (Tyrosyl DNA phosphodiesterase-1) = Hydrolyses the 3’-phosphotyrosyl bond that links Top1 to a DNA strand break
  □ TDP2 (Other names TTRAP/EAPII) = Processes both 5′- and 3′-phosphotyrosyl termini, but ~ 50-fold more active on 5’. TDP2-dependent non-homologous end-joining protects against topoisomerase II-induced DNA breaks and genome instability in cells and in vivo.
  □ Pathways of double strand break repair (NHEJ, Homologous RR)
  □ Alteration to signalling pathways that link DNA damage detection to apoptosis