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topoisomerases simplified
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  2. 2. • The recognition of a base sequence requires the local separation of complementary polynucleotide strands. • Naturally occurring DNAs  Negative supercoiling • The supercoiling of DNA is controlled by a remarkable group of enzymes known as DNA topoisomerases (or simply topoisomerases).
  3. 3. There are two classes of topoisomerases: TYPE I TOPOISOMERASES • James Wang in 1971 • Act by creating transient ss breaks in DNA • Type I topoisomerases are denoted by odd Roman numerals (e.g. topoisomerase I, III, etc.). • Subclasses (IA, IB, IC) • Based on AA Sequence & • MOA TYPE II TOPOISOMERASES • Martin Gellert in 1976 • act by creating transient ds breaks in DNA (ATP used) • Type II topoisomerases are denoted by even Roman numerals (e.g., topoisomerase II, IV, etc.). • Subclasses (IIA, IIB) • Based on AA Sequence
  4. 4. Type I topoisomerases • All cells • Relax only -ve supercoiled DNA • E.Coli • Topoisomerase I (ω protein) • Topoisomerase III • Eubacteria & Archaea • Reverse Gyrase • Strand Passage Mech. • Forms 5’-P-Tyr intermediates TYPE IA TYPE IB TYPE IC • Eukaryotes & many prokaryotes (E.Coli) • Relax both -ve & +ve supercoils • Human • Topoisomerase I • Controlled Rotation Mechanism • Forms 3’-P-Tyr intermediates • Archaea • Archaea • Topoisomerase V • Controlled Rotation Mechanism • Forms 3’-P-Tyr intermediates
  5. 5. • Many hyper thermophiles (both eubacteria and archaea) • Type IA topoisomerase known as reverse Gyrase induces positive supercoiling in DNA through the ATP-driven action • (cf Gyrase  Induce negative supercoiling) • This suggests that positive supercoiling, which tightens the DNA double helix, protects DNA from thermal denaturation.
  6. 6. Mechanism Of Action • Apparently the enzyme operates by • cutting a single strand, • passing a single-strand loop through the resulting gap, and • then resealing the break, • thereby twisting double helical DNA by one turn.
  7. 7. Type IA Topoisomerase Functions via a Strand Passage Mechanism
  8. 8. • The protein recognizes a ss region of the DNA (red strand) and binds it in its binding groove(Yellow). • This is followed by (or occurs simultaneously) with the opening of a gap between domains I and III. • Curiously, this single-stranded DNA assumes a B-DNA-like conformation even though its complementary strand would be sterically excluded from the groove.
  9. 9. • The DNA is cleaved • Newly formed 5’ end becoming covalently linked to the active site Tyr and • The newly formed 3’ end remaining tightly but noncovalently bound in the binding groove.
  10. 10. • The unbroken (green) strand is passed through the opening or gate formed by the cleaved (red) strand to enter the protein’s central hole.
  11. 11. • The unbroken strand is trapped by the partial closing of the gap.
  12. 12. The two cleaved ends of the red strand are re-joined in what is probably a reversal of the cleavage reaction.
  13. 13. • The gap between domains I and III re-opens to permit the escape of the red strand, yielding the reaction product in which the green strand has been passed through a transient break in the red strand.
  14. 14. • The enzyme returns to its initial state.
  15. 15. Support of this strand passage mechanism • The denaturation of type IA enzyme that has been incubated with single-stranded circular DNA yields a linear DNA that has its 5’-terminal phosphoryl group linked to the enzyme via a phosphoTyr diester linkage. • By forming such covalent enzyme–DNA intermediates, the free energy of the cleaved phosphodiester bond is preserved, so that no energy input is required to reseal the nick.
  16. 16. Type IB Topoisomerase Functions via a Controlled Rotation Mechanism
  17. 17. Binding Cleavage Religation & Release
  18. 18. • Topoisomerase I relaxes DNA supercoils by permitting the cleaved duplex DNA’s loosely held downstream segment to rotate relative to the tightly held upstream segment. • This rotation can only occur about the sugar– phosphate bonds in the uncleaved strand (α, β, ε, and ζ) that are opposite the cleavage site because the cleavage frees these bonds to rotate.
  19. 19. Type II topoisomerases • Bacteria (A2B2 heterotetramers) • DNA Gyrase (Just Gyrase) • Topoisomerase IV • Eukaryotic (AB homodimer) • Topoisomerase II TYPE IIA TYPE IIB • Only one member that occurs in Archaea • Topoisomerase VI Note: • Gyrase  Generates negative supercoils. [cf Reverse Gyrase(Type IA)  Generates positive supercoils] • All other topoisomerases only relax supercoils.
  20. 20. Gyrase acts by cutting both strands of a duplex, passing the duplex through the break, and resealing it. So, the linking numbers differ by increments of 2 rather than 1, as occurs with type I topoisomerases.
  21. 21. Strand Passage Mechanism ATPase Domain Breakage/Reunion Domain
  22. 22. Strand Passage Mechanism DNA duplex to be cleaved (G-segment, G for Gate) binds in the groove across the top of the cleavage/reunion domain & leads to conformational changes drawn in 2. (Interestingly, the DNA between the two cleavage sites is essentially in the A form.)
  23. 23. Strand Passage Mechanism The binding of ‘ATP and a T- segment’ Induces a series of conformational changes. • The G-segment is cleaved and the resulting two fragments are spread apart by at least 20 Å. • The ATPase domains dimerize, and • The T-segment (T for transported) is transported through the break in G- segment & breakage/reunion domain’s upper gate into the central hole  thereby incrementing the DNA’s linking number by 2
  24. 24. Strand Passage Mechanism The DNA transport step is shown as proceeding through the hypothetical intermediate in square brackets. The G segments are then resealed and the T- segment is released through the lower gate.
  25. 25. Strand Passage Mechanism This gate then closes as the ATP is hydrolyzed and the resulting ADP and Pi released to yield the enzyme in its starting state (2).
  26. 26. Topoisomerase Inhibitors Are Effective Antibiotics and Cancer Chemotherapy Agents ANTIBIOTICS (SPECIFICALLY INHIBIT GYRASE AND ARE THEREFORE ANTIBIOTICS) • Specifically inhibit Gyrase and are therefore antibiotics • Novobiocin (Coumarin derivatives), and • ciprofloxacin (quinolone derivatives) • Active against Gram –ve bacteria. CANCER CHEMOTHERAPEUTIC AGENTS • Inhibit eukaryotic type IIA topoisomerases and are therefore widely used in cancer chemotherapy • doxorubicin (AKA Adriamycin; a product of Streptomyces peucetius) and • Etoposide (a synthetic derivative)