CA García Sepúlveda MD PhD

1. Transposons CA García Sepúlveda MD PhD Laboratorio de Genómica Viral y HumanaFacultad de Medicina, Universidad Autónoma de San Luis Potosí

2. Session #25-26 TransposonsIntroduction • Genomes evolve both by rearranging existing sequences and by acquiring new sequences. • Rearrangements are sponsored by internal genomic events. • Unequal recombination (non-recirpocal) results from mispairing by the cellular mechanisms for homologous recombination. • Results in duplication or rearrangement of loci (Clusters & repeats). • Duplication of sequences within a genome gives rise to further duplication.

3. Session #25-26 TransposonsIntroduction • Genomes evolve both by rearranging existing sequences and by acquiring new sequences. • Results from the ability of vectors to carry information between genomes. Plasmids move by conjugation. Extrachromosomal elements move information horizontally by mediating the transfer of short lengths of genetic material.

4. Session #25-26 TransposonsIntroduction • Genomes evolve both by rearranging existing sequences and by acquiring new sequences. • Results from the ability of vectors to carry information between genomes. Phages spread by infection. Both plasmids and phages occasionally transfer host genes along with their own replicon.

5. Session #25-26 TransposonsIntroduction • Genomes evolve both by rearranging existing sequences and by acquiring new sequences. • Results from the ability of vectors to carry information between genomes. • Direct transfer of DNA occurs between some bacteria by means of transformation.

6. Session #25-26 TransposonsIntroduction • Genomes evolve both by rearranging existing sequences and by acquiring new sequences. • Results from the ability of vectors to carry information between genomes. In eukaryotes, some viruses (notably the retroviruses) can transfer genetic information during an infective cycle.

7. Session #25-26 TransposonsIntroduction • Genomes evolve both by rearranging existing sequences and by acquiring new sequences. • Another major cause of variation is provided by transposable elements or transposons: • these are discrete sequences in the genome that are mobile & able to transport themselves to other locations within the genome. • Found in both eukaryotes & prokaryotes. • Selfish DNA with the sole purpose of autoreplication. Visit: http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter13/animation_quiz_5.html

8. Session #25-26 TransposonsIntroduction • Relationship of the transposon to the genome resembles that of a parasite with its host. • The propagation of an element by transposition is balanced by the harm done if a transposition event inactivates a necessary gene. • Any transposition event conferring a selective advantagewill lead to preferential survival of the genome harboring the transposon !

9. Session #25-26 TransposonsTransposons • Transposons do not utilize an independent form (such as virus or plasmid DNA). • Move directly from one site of the genome to another. • Unlike other processes involved in genome restructuring, transposition does not rely on homology between donor and recipient sites. • Sometimes transfer contiguous host sequences to new sites elsewhere within the same genome as they move. • They are an internal counterpart to the vectors that can transport sequences from one genome to another.

10. Session #25-26 TransposonsTransposons • Transposable elements can promote rearrangements of the genome, directly or indirectly: • Directly: The transposition event itself may cause deletions or inversions or lead to the movement of a host sequence to a new location. • Indirectly: Transposon sequences serve as substrates for cellular recombination systems by functioning as "portable regions of homology"; • two copies of a transposon at different locations (even on different chromosomes) may provide sites for reciprocal recombination resulting in deletions, insertions, inversions, or translocations.

11. Session #25-26 TransposonsTransposons • They may provide the major source of mutations in the genome! • Two general classes of transposons: • DNA transposons Exist as sequences of DNA coding for proteins that are able directly to manipulate DNA so as to propagate themselves within the genome. • RNA transposonsThey are related to retroviruses and move as a consequence of their ability to make DNA copies of their RNA transcripts, the DNA copies then become integrated at new sites in the genome.

12. Session #25-26 TransposonsTransposons • Transposons carry gene(s) that code for the enzyme activities required for their own transposition. • However, it may also require host machinery (DNA pol or DNA gyrase).

13. Session #25-26 TransposonsDiscovery • Transposable elements were first identified in the form of spontaneous insertions in bacterial operons. • Such an insertion prevents transcription and/or translation of the gene in which it is inserted. • The first transposons that were discovered were simple and called insertion sequences (IS). • Each type is given the prefix IS, followed by a number that identifies the type. The original classes were IS1-4, more classes have been discovered since. • Insertion into a particular site described with a double colon: l::IS1 = An IS1 element inserted into phage lambda.

14. Session #25-26 TransposonsInsertion Sequences (IS) • IS elements are normal constituents of bacterial chromosomes & plasmids. • A standard strain of E. coli contains <10 copies of the more common IS elements. • The IS elements code onlyfor the proteins needed to sponsor its owntransposition. • Each IS element is different in sequence, but there are common organizational features.

15. Session #25-26 TransposonsInsertion Sequences (IS) • An IS element ends in short inverted terminal repeats which are not identical but closely related. • Inverted repeats recognized by TRANSPOSASE. • Ensure that the same sequence is encountered proceeding toward theelement from any direction. • Inverted repeat recognition is common to transposition events sponsored by all transposons. • Cis-acting mutations of these ends prevent transposition.

16. Session #25-26 TransposonsInsertion Sequences (IS) • When an IS element transposes, a sequence of host DNA at the site of insertion is duplicated: DIRECT REPEATS. • IS DNA is always flanked by very short direct repeats with the samedirection. • Pre-transposition genomic sequencesexhibit only one of these “repeats” (i.e.: ATGCA). • Post-transposition sequence will have this sequence duplicated and flanking the transposon sequence.

17. Session #25-26 TransposonsInsertion Sequences (IS) • IS display a characteristic structure in which its ends possess inverted terminal repeats while the adjacent ends of the flanking host DNA possess short direct repeats. • This type of organization is taken to be diagnostic and suggest that the sequence originated in a transposition event. • IS elements insert at a variety of sites within host DNA, some show preference for particular hotspots

18. Session #25-26 TransposonsInsertion Sequences (IS) • All IS elements (except IS1) contain a single long coding region for transposase starting after the inverted repeat at one end and terminating before or within the inverted repeat at the other end. • IS1 is more complex, it employs two separate reading frames. • Frequency of transposition varies amongst the different elements.

19. Session #25-26 TransposonsInsertion Sequences (IS) • What would happen if an IS transposed near the original position... or if two IS sequences were separated by genomic DNA? • These transposons are called COMPOSITE TRANSPOSONS (Tn).

20. Session #25-26 TransposonsComposite Transposons (Tn) • Code for more than proteins involved in transposition. • A central genomic “core” flanked by two IS.

21. Session #25-26 TransposonsComposite Transposons (Tn) • Central region discovered initially as carrying drug markers or drug resistance traits. • IS modules ("arms") may have same or inverted orientations (most common).

22. Session #25-26 TransposonsComposite Transposons (Tn) • Central region discovered initially as carrying drug markers or drug resistance traits. • IS modules ("arms") may have same or inverted orientations (most common). • In some cases the modules are identical • Tn9 (direct repeats of IS1) • Tn903 (inverted repeats of IS903). In other cases, the modules are only closely related (Tn10, Tn5).

23. Session #25-26 TransposonsComposite Transposons (Tn) • A functional IS module can transpose either itself or the entire transposon. • Either identical module of a composite transposon can sponsor movement (IS10L or IS10R). • In transposons with different modules transposition might depend entirely or principally on one of the modules (Tn10 or Tn5). • What is responsible for transposing a composite transposon instead of just the individual module?

24. Session #25-26 TransposonsComposite Transposons (Tn) • 1.- IS vs Tn equally feasible & useful from a “selfish point of view”. • 2.- Selective pressure.

25. Session #25-26 TransposonsComposite Transposons (Tn) • 1.- IS vs Tn equally feasible & useful from a “selfish point of view”. • 2.- Selective pressure. • Two IS elements can transpose any sequence residing between them just as well as themselves. • Exemplified by transposons in bacteria where the two modules can be considered to flank either the tetR gene of the original Tn10 or the genomic sequence in the other part of the circle.

26. Session #25-26 TransposonsComposite Transposons (Tn) • 1.- IS vs Tn equally feasible & useful from a “selfish point of view”. • 2.- Selective pressure. • Selection for the trait(s) carried in the central region. • An IS10 module mobilizes an order of magnitude more frequently than Tn10. • But Tn10 is held together by selection for tetR; so that under selective conditions, the relative frequency of intact Tn10 transposition is higher.

27. Session #25-26 TransposonsComposite Transposons (Tn) • The insertion of a transposon into a new site consists of: • Making staggered breaks in the target DNA • Joining the transposon to the protruding single-stranded ends • Filling in the gaps. The stagger between the cuts determines the length of the direct repeats and reflects the geometry of the enzyme involved in cutting target DNA.

28. Session #25-26 TransposonsComposite Transposons (Tn) • The insertion of a transposon into a new site consists of: • Making staggered breaks in the target DNA • Joining the transposon to the protruding single-stranded ends • Filling in the gaps.

29. Session #25-26 TransposonsComposite Transposons (Tn) • The insertion of a transposon into a new site consists of: • Making staggered breaks in the target DNA • Joining the transposon to the protruding single-stranded ends • Filling in the gaps. The generation and filling of the staggered ends explain the direct repeats of target DNA at the site of insertion. The use of staggered ends is common to all transposons!

30. Session #25-26 TransposonsComposite Transposons (Tn) • Three different types of mechanism by which a transposon moves: • Replicative Transposition • Common Non-replicative Transposition • Conservative Non-replicative Transposition

31. Session #25-26 TransposonsComposite Transposons (Tn) • Three different types of mechanism by which a transposon moves: • Replicative Transposition • The element is duplicated during the reaction, so that the transposing entity is a copy of the original element. • One copy remains at the original site, while the other inserts at the new site. • Transposition is accompanied by an increase in the number of copies.

32. Session #25-26 TransposonsComposite Transposons (Tn) • Three different types of mechanism by which a transposon moves: • Replicative Transposition • Involves two types of enzymatic activity: • Transposase that acts on the ends of the original transposon. • Resolvase that acts on the duplicated copies.

33. Session #25-26 TransposonsComposite Transposons (Tn) • Three different types of mechanism by which a transposon moves: • Non-Replicative Transposition • Two types: • Common Non-replicative Transposition. • Conservative transposition (now known as Episomal transposition or simply Episome).

34. Session #25-26 TransposonsComposite Transposons (Tn) • Three different types of mechanism by which a transposon moves: • Non-Replicative Transposition • The transposing element moves as a physical entity directly from one site to another • without copies • without change • Requires only a Transposase • Tn10 & Tn5

35. Session #25-26 TransposonsComposite Transposons (Tn) • Three different types of mechanism by which a transposon moves: • Common Non-Replicative Transposition • Disregards double strand cleavage of genomic DNA from which it originated. • Relies on host repair mechanisms to repair double strand breaks.

36. Session #25-26 TransposonsComposite Transposons (Tn) • Three different types of mechanism by which a transposon moves: • Conservative Non-replicative Transposition • the element is excised from the donor site and inserted into a target site by a series of events in which every nucleotide bond is conserved. • Transposon looking after the health of its host. • Mechanism of lambda Phage integration.

37. Session #25-26 TransposonsComposite Transposons (Tn) • Three different types of mechanism by which a transposon moves: • Conservative Non-replicative Transposition • The elements that use this mechanism are large, and can mediate transfer of donor DNA from one bacterium to another. • Although originaly transposons, more appropriate name is Episomes.

38. Session #25-26 TransposonsComposite Transposons (Tn) • Transposons may use only one or different types of pathway for transposition. • Basic reactions involved in all classes of transposition event: • The ends of the transposon are disconnected from the donor DNA by cleavage reactions that generate 3’-OH ends. • The exposed ends are joined to the target DNA by trans-esterification in which the 3’-OH end directly attacks the target DNA.

39. Session #25-26 TransposonsComposite Transposons (Tn) • Reactions take place within nucleoprotein complex (enzymes and both ends of the transposon). • Target site is chosen by transposase (random vs. specificity) • for a consensus sequence, • for a structure, such as bent DNA, • for inactive regions of the chromosome.

40. Session #25-26 TransposonsComposite Transposon DNA rearrangements • Transposons promote other types of DNA rearrangements. • Some of these events are consequences of the multiple copies generated (gene duplications). • Others represent alternative outcomes of the transposition mechanism.

41. Session #25-26 TransposonsComposite Transposon DNA rearrangements • Rearrangements of host DNA may result when a transposon inserts a copy at a second site near its original location. • Host (or transposon) systems may undertake reciprocal recombination between the two copies of the transposon. • The consequences are determined by whether the repeats are the same or in inverted orientation.

42. Session #25-26 TransposonsComposite Transposon DNA rearrangements • Recombination between direct repeats will delete the material between them. • The intervening region is excised as a circle of DNA (which is lost from the cell). • The chromosome retains a single copy of the direct repeat. • A recombination between the directly repeated IS1 modules of the composite transposon Tn9 would replace the transposon with a single IS1 module… This doesn’t normally happen! Why?

43. Session #25-26 TransposonsComposite Transposon DNA rearrangements • Excision is not supported nor coded by transposons themselves. • Mechanism is not known. • Excision is RecA-independent. • Might occur by some cellular mechanism that generates spontaneous deletions between closely spaced repeated sequences.

44. Session #25-26 TransposonsComposite Transposon DNA rearrangements • Reciprocal recombination between a pair of inverted repeats. • The region betweenthe repeatsbecomesinverted. • The repeats themselves remain available to sponsor further inversions. • A composite transposon whose modules are inverted is a stable component of the genome, although the direction of the central region with regard to the modules could be inverted by recombination. • Direction influences transcription and translation!

45. Session #25-26 TransposonsComposite Transposon DNA rearrangements • Reciprocal recombination between a pair of inverted repeats. • The region betweenthe repeatsbecomesinverted. • The repeats themselves remain available to sponsor further inversions. • A composite transposon whose modules are inverted is a stable component of the genome, although the direction of the central region with regard to the modules could be inverted by recombination. • Direction influences transcription and translation!

46. Session #25-26 TransposonsTransposition Intermediates • Many mobile DNA elements transpose from one chromosomal location to another by a fundamentally similar mechanism. • IS elements • Prokaryotic & eukaryotic transposons • Bacteriophage Mu. • Retroviral RNA integration. • The first stages of immunoglobulin recombination.

47. Session #25-26 TransposonsTransposition Intermediates • Transposon is nicked at both ends. • Target sequence is nicked at both ends.

48. Session #25-26 TransposonsTransposition Intermediates • The nicked ends are joined crosswise to generate a covalent connection between the transposon and the target. • The two ends of the transposon are brought together in this process. • FIGURE NOTE: for simplicity in following the cleavages, the synapsis stage is shown after cleavage, but actually occurs BEFORE CLEAVAGE.

49. Session #25-26 TransposonsTransposition Intermediates • A more realistic image • The strand transfer complex in which the transposon is connected to the target site through one strand at each end. • The next step of the reaction differs and determines the type of transposition.

50. Session #25-26 TransposonsTransposition Intermediates • A more realistic image • The strand transfer complex can be a target for replication (leading to replicative transposition). • Or the strand transfer complex can be a target for repair (non-replicative transposition).