Transposons & Mechanisms of Transposition

Krystine Garcia, Tao Jing, Alexander Meyers

Genomic Distribution98.5% of human genome non-

coding25% non-repeat spacer DNARepetitious DNA

-Tandemly repeated genes-Sattelite DNA (6%)

Interspaced repeats

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.

Transposable ElementsBegan as symbiont DNATransposons provide a

mechanism for bringing about DNA rearrangements throughout evolution

Adjacent DNA sequences sometimes mobilized

Transposons, retrotransposons

Transposons and RetrotransposonsTransposons excise themselves

and move to another locationRetrotransposons duplicate and

reintegrate themselves

IS: Insertion Sequence

IS: Insertion Sequence

TransposaseCis-acting enzymeIs coded for in

transposonCuts the IS at

inverted repeatsTransmits 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

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

Autonomous & NonautonomousAutonomous (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

3 principle classes of transposons:1. DNA transposons: move using cut and paste or replicative mechanism

2. Virus-like retrotransposons (aka long terminal repeat [LTR] retrotransposons): RNA intermediate, includes retroviruses

3. Poly-A retrotransposons (aka nonviral retrotransposons): RNA intermediate

Cut and paste mechanism of transposition:

Nonreplicative1. Transposase (usually 2 or 4 subunits)

binds terminal inverted repeats2. Brings 2 ends together stable protein

complex called synaptic complex or transpososome

3. 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

4. Other DNA strands cut by various mechanisms transposon excised

Cut and paste mechanism of transposition:

5. 3’-OH ends of transposon DNA attack DNA phosphodiester bonds at site of new insertion (target DNA)

6. Nicks introduced in other target DNA strands few nucleotides apart transposon joined via reaction called DNA strand transfer

7. Few nucleotides between nicks leaves small ss gaps filled in by host DNA repair polymerase small target site duplications on either side transposon

8. DNA ligase seals final nicks9. Ds break where transposon left

repaired by homologous recombination

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

Nontransferred strand cleavage:

Replicative transposition:Transposon DNA replicated during

each round of transposition1. Transposase assembles on each end of

transposon to form transpososome2. 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. 3’-OH joined to target DNA by strand transfer reaction (same mechanism as cut-and-paste) intermediate is double branched DNA molecule

Replicative transposition:4. 3’ ends transposon covalenty linked to

target DNA, but 5’ ends still linked to old flanking DNA

5. 2 branches like replication forks, DNA replication proteins assemble at these forks, 3’-OH serves as primer

6. 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

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

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

Virus-like retrotransposons and retroviruses:1. Retrotransposon DNA transcribed into

RNA by host RNAP (transcription starts at promoter within LTR)

2. RNA reverse-transcribed (by RT) RNA:DNA dsDNA (cDNA)

3. 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)

4. Integrase performs strand transfer reaction to insert 3’ ends into target DNA

5. Gap fill and ligation by host proteins

Transposon Excision

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