Transposons & Mechanisms of Transposition

1. Transposons & Mechanisms of Transposition Krystine Garcia, Tao Jing, Alexander Meyers

2. Genomic Distribution • 98.5% of human genome non-coding • 25% non-repeat spacer DNA • Repetitious DNA-Tandemly repeated genes-Sattelite DNA (6%) • Interspaced repeats

3. Transposable Elements Interspaced repeats have the capability to “move around” in the genome, and are thus referred to as transposable elements. Transposition in germ cells are passed down to progeny resulting in an accumulation in the genome.

4. Transposable Elements • Began as symbiont DNA • Transposons provide a mechanism for bringing about DNA rearrangements throughout evolution • Adjacent DNA sequences sometimes mobilized • Transposons, retrotransposons

7. Transposons and Retrotransposons • Transposons excise themselves and move to another location • Retrotransposons duplicate and reintegrate themselves

8. IS: Insertion Sequence

9. IS: Insertion Sequence

10. Transposase • Cis-acting enzyme • Is coded for in transposon • Cuts the IS at inverted repeats • Transmits it to another part of the DNA at a target sequence • Multiple types of transposase http://www.rcsb.org/pdb/101/motm.do?momID=84

11. Transposase Regulation • Frequency of transposition is regulated by transposase regulation • Not all transposase genes are transcribed http://www.rcsb.org/pdb/101/motm.do?momID=84

12. Autonomous & Nonautonomous • Autonomous (activator elements) are very similar to bacterial IS elements in structure and function • Nonautonomous (dissociation elements) lack transposase gene • Cannot move by themselves • Must have a cis transposable element with transposase gene to move

13. 3 principle classes of transposons: • DNA transposons: move using cut and paste or replicative mechanism • Virus-like retrotransposons (aka long terminal repeat [LTR] retrotransposons): RNA intermediate, includes retroviruses • Poly-A retrotransposons (aka nonviral retrotransposons): RNA intermediate

14. Cut and paste mechanism of transposition: • Nonreplicative • Transposase (usually 2 or 4 subunits) binds terminal inverted repeats • Brings 2 ends together  stable protein complex called synaptic complex or transpososome • Transposase cleaves one DNA strand at each end at junction between transposon DNA and host DNA  transposon sequence terminates with free 3’-OH groups at each end • Other DNA strands cut by various mechanisms  transposon excised

15. Cut and paste mechanism of transposition: • 3’-OH ends of transposon DNA attack DNA phosphodiester bonds at site of new insertion (target DNA) • Nicks introduced in other target DNA strands few nucleotides apart  transposon joined via reaction called DNA strand transfer • Few nucleotides between nicks leaves small ss gaps  filled in by host DNA repair polymerase  small target site duplications on either side transposon • DNA ligase seals final nicks • Ds break where transposon left repaired by homologous recombination

16. Nontransferred strand cleavage: • Nontransferred strands= 5’ ends of transposon (ends not covalently linked to target DNA during strand transfer) • Cleavage can use enzyme other than transposase: • Tn7 encodes specific protein called TnsA with structure similar to restriction enzyme – works with transposase to cleave nontransferred strands • Cleavage can be performed by transposase: • Tn5 and Tn10 form DNA hairpin (3’-OH attacks its complementing strand) to cause nicks  hairpin opened by transposase  3’-OH’s join to target DNA via strand transfer • Hermes forms DNA hairpins in target DNA

17. Nontransferred strand cleavage:

18. Replicative transposition: • Transposon DNA replicated during each round of transposition • Transposase assembles on each end of transposon to form transpososome • Transposase introduces nicks at junctions between transposon and flanking host DNA  generates 3’-OH ends on transposon (but transposon NOT excised from flanking DNA) • 3’-OH joined to target DNA by strand transfer reaction (same mechanism as cut-and-paste)  intermediate is double branched DNA molecule

19. Replicative transposition: • 3’ ends transposon covalenty linked to target DNA, but 5’ ends still linked to old flanking DNA • 2 branches like replication forks, DNA replication proteins assemble at these forks, 3’-OH serves as primer • Replication proceeds through transposon and stops at 2nd fork  2 copies of transposon flanked by short target site duplications • Frequently causes chromosomal inversions and deletions detrimental to host

20. Virus-like retrotransposons and retroviruses: • Carry inverted terminal repeats (recombination sequences) embedded within longer direct repeat sequences (aka long terminal repeats [LTR]) • Encode 2 proteins needed for mobility: integrase (transposase) and reverse transcriptase

21. Virus-like retrotransposons and retroviruses: • Reverse transcriptase: Enzyme that uses RNA template to synthesize DNA • Retrovirus: genome packaged into viral particle, leaves host cell, infects new cell • Retrotransposon: can only move to new DNA sites within cell

22. Virus-like retrotransposons and retroviruses: • Retrotransposon DNA transcribed into RNA by host RNAP (transcription starts at promoter within LTR) • RNA reverse-transcribed (by RT)  RNA:DNA  dsDNA (cDNA) • Integrase (transposase) recognizes and binds ends of cDNA then cleaves few nucleotides off 3’ end of each strand (just like cleavage step of DNA transposons) • Integrase performs strand transfer reaction to insert 3’ ends into target DNA • Gap fill and ligation by host proteins

24. Transposon Excision

25. Plant genomes are rich in transposons: • Barbara McClintock discovered transposons in the late 1940’s • Maize color varigation due to chromosome breakage by transposition • Snapdragons: size of white patches related to frequency of transposition