Topoisomerases Flashcards
what is topology and how does it relate to DNA
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.
What do topoisomerase do ?
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
What is difference between the classes of topoisomerase enzymes
type 1 = one a single DNA is cut
type 2 = both strands are cut
What is the topological constraints on DNA
○ 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
What are human topoisomerase function
○ 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
How do topoisomerase II poisons work and what are the current uses
- 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
What is etoposide
Top II poison, weak anti-tumour activity
- created etoposide then teniposide
what are the functions of topoisomerase II poisons
- 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.
how are cleavable complexes formed in the cell detected
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)
What are anthracyclines
- 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
Why cant patients be given camptothecin
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
what is Irinotecan
Topoisomerase I poison
a pro-drug which forms SN-38 (active form) by carboxylesterases CES1/CES2
how is SN-38 metabolised
primarily in the liver to the inactive SN-38 glucuronide by uridine diphosphate glucuronosyltransferase (UGT1A1)
excreted in bile
why is there a large variability in irinotecan response in patients
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
what is the function of irinotecan and what is the mechanisms
Breaks single strand of DNA, forms covalent bonds
Allows rotation of one strand around another
Camptothecin forms a cleavable complex
-> Leads to downstream effects
How does topoisomerase enzyme cause cytotoxicity
○ 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
What happens to cells after topoisomerase
DNA damage -> Delay of cell cycle progression (repair/removal of damage)
either
- cell division not possible (mitotic catastrophe)
- cell survives
- Apoptosis triggered by damage
T/F topoisomerase poisons are specific
False
non-specific hence sever toxic side effects
Why do cytotoxic drugs preferentially attack tumours
- 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
What are the resistance mechanism involved in topoisomerase poison treatment
- less access to target (less drug in cell)
- Altered target or less target
- altered response to DNA damage
How does less access to target causes resistance form topoisomerase poisons
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
How does altered/less target causes resistance form topoisomerase poisons
□ 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
How does an altered response to DNA damage cause resistance to topoisomerase poisons
- 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
