Lecture 8: Nucleic Acid Structure II Flashcards
Topoisomerases
-catalyze concerted breakage and rejoining of DNA strands
-control formation of superhelical DNA
Topoisomerase enzyme function
-break and rejoin DNA strands
-need to alter topological status of DNA by changing the linking number and therefore the number of supercoils
Topoisomerase biological role
-control formation of superhelical DNA
-resolve winding problem from replication and transcription = altered DNA topology
Topology control
-cells need for survival
-DNA topology altered during replication and translation
No topology control
-accumulation of supercoils
-hinders melting of DNA duplex
-hinders movement of protein machinery along DNA
Human topoisomerases
Top1 and Top2
-targets for anticancer drugs
Eukaryotic topoisomerases
-do not introduce supercoils
-only relaxes them
prokaryotic topoisomerase
can introduce negative supercoils
Top1
-cuts only ONE strand of DNA
-cut strand rotates around intact
-no energy needed
-removes +/- supercoiling
-bacterial and eukaryotic enzymes
-change LK by 1 ?
Top2
-cuts both strands of DNA
-requires ATP
-moves another dsDNA segment through opening in cut strand
-changes linking number by 2
-bacterial and eukaryotic enzymes
-gyrase
Gyrase
-Top2
-only bacteria
-adds negative supercoils
Supercoiling of linear DNA
-generated by protein complexes translocating along DNA
-anchoring nuclear proteins create closed topological domains
Where is DNA overwound
-supercoiling ahead of replication fork
-jumprope
Where is DNA underwound
-DNA before replication fork
Top1 mechanism
- Cleaves one DNA strand
- Forms a transient covalent bond between tyrosine residue and phosphoryl group at cleavage site
- DNA relaxes by controlled rotation of the free end
- Seal gap by re-ligating cleaved strand
-slide 7 pic nvm its wrong
Top1 tyrosine residue phosphoryl group bond
-different top1 enzymes form enzyme link as either 3’ or 5’ -phosphotyrosine
Controlled rotation of free end
-relaxes DNA
-free end swivels around intact strand
-add or subtract turns depending on substrate DNA
Underwound
Top1 adds turns
Overwound
Top1 removes turns
Top2 mechanism
-needs ATP hydrolysis
1. breaks 2 strands of DNA
2. passes duplex DNA through gap and reseal gap
-eukaryotic relaxes supercoil
-prokaryotic add supercoil
Topoisomerase inhibitors
-Antineoplastic and anitmicrobial agents
-anticancer targeting
-trap intermediate complex of enzyme and DNA
-degradation of DNA, addition of mutations, or inhibition of transcription replication and repair
TOP1 drugs (Camptothecin Class)
-irinotecan and topotecan
-derivatives of camptothecin (tree bark)
-inhibit re-ligation step
-traps Top1-DNA complex
-replication fork hits complex = ds break = apoptosis
Topotecan
ovarian and cervical cancer
SLIDE 8 is remove turn
idk why
minus one
negative supercoiling
underwound
irinotecan
-prodrug converted to active DN-38
-combo therapy for colon or rectum carcinomas
indenoisoquinoline
-another Top1 drug class
-shamless Dr Cushman plug i think
Drug action of Top1 drugs
-binds covalent intermediate and stops religation
-crystal structures define interactions
-camp vs indeno interactions and residence time
G-quadraplex structure
-4 guanines in plane with central cation
-sequence variation in structure
-top structure same
-different sequences have alternative strand directions (bottom)
Topology of promoter regions
-changed by singlestranded structures generated from DNA transcription
G-Quadraplex function
-telomer extension
-genome stability
-regulation of expression/replication/translation
-sensor of oxidative DNA damage
G-Quadraplex
-found in human promoter regions (esp of highly transcribed genes like oncogene MYC)
-formation and stabilization should stall transcription machinery = inhibit transcription
TmPyP4 porphyrin
-binds to G-quadraplex of MYC promoter
-inhibits MYC transcription
-suppression of gene expression
-anticancer activity
