DNA Mobility and Transposition (PDF)

DNA Mobility Brendan Cormack Mobile Genetic Elements Common to all three domains of life: prokaryotes, archaea and eukaryotes Drivers of genomic plasticity, with impact on genome structure, function and evolution For some elements mobility occurs by way of an RNA int...

DNA Mobility Brendan Cormack Mobile Genetic Elements Common to all three domains of life: prokaryotes, archaea and eukaryotes Drivers of genomic plasticity, with impact on genome structure, function and evolution For some elements mobility occurs by way of an RNA intermediate, for others only DNA is involved Mobility can be stochastic or programmed Mobile elements have been harnessed for experimental mutagenesis or gene transfer Transposition – the movement of a discrete DNA element into diverse target sites Movement is mediated by proteins specific to a particular element Transposition is accompanied by duplication of target sequence – “TSD” Transposable elements are potent sources of genetic diversity Disruption of genes Mobilization of genes within and among chromosomes Alteration of gene expression by placing TE regulatory signals near host genes (promoters, enhancers, splice sites, polyA sites, etc) Substrates for homologous recombination Genomes, through the actions of transposable elements, are highly dynamic structures Transposons reveal themselves by generating mutations Barbara McClintock – controlling elements (i.e., transposons) in maize move to different positions, thus influencing pigment expression Mutation also led to identification of the first active human transposon Haig Kazazian (Johns Hopkins) – two unrelated boys (of 112 total) had insertions in Factor VIII gene, not present in their parents Inserted sequences – non-LTR LINE elements (see below) Many human diseases now known to be caused by TE insertion Fraction of de novo mutations caused by transposon insertion varies 0.3% in humans (TE comprise about 45% of the genome) 10% in mice (TE comprise about 38% of the genome) In humans and mice, 1 insertion every 20 – 100 live births and most of these are inconsequential because most genome is noncoding >50% in Drosophila (TE comprise about 5.5% of the genome) Classification of transposable elements TEs can be grouped according to several criteria Element structure Transposase structure Mechanism of transposition Effects of transposition on the donor site Major groups according to element structure DNA-only intermediates RNA intermediates Long Terminal Repeat (LTR) elements Retroviruses Retroviral-like elements non-LTR elements Genome components in a variety of organisms. DNA-only transposons Transposase acts on terminal inverted repeats (TIR) “Cut and paste” or replicative (“nick and paste”) mechanisms of movement Non-autonomous elements rely on exogenous transposase Long terminal repeat (LTR) elements Transposition cycle involves alternation between RNA and DNA copies of the element LTRs support the conversion of RNA copy to cDNA, which is integrated into the host genome LTRs subsequently support transcription of the integrated DNA copy into an RNA copy Transposition proteins encoded by pol: reverse transcriptase and integrase (transposase) Retroviruses – extracellular transmission from cell to cell (env encodes surface glycoprotein) Retroviral-like elements – strictly intracellular Non-LTR elements LINE (Long Interspersed Element) – 21% of human genome Autonomous Transposition proteins: ORF1 – RNA chaperone ORF2 – RT and endonuclease SINE (Short Interspersed Element) – 13% of human genome Non-autonomous Dependent on LINE proteins for transposition A and B – RNA polymerase III promoter DNA transposon superfamilies Bacterial IS4 (includes Tn5, Tn10) Mu-like (Mu, Tn7) Tn3, gd Eukaryotic Mutator (maize) P element (D. melanogaster) Tc1/mariner (Sleeping Beauty) hAT piggyBac DNA-only cut and paste elements Bacterial insertion sequences – IS elements Inverted repeats flanking transposase gene Composite bacterial transposons IS elements flank other genes (e.g., drug resistance) DNA-only cut and paste transposition strategy Transposase binding, pairing to activate DNA cleavage Excision to expose 3’OH at each end Target joining (strand transfer) by nucleophilic attack of 3’OH on phosphodiester bonds in target DNA. Gap repair results in TSD. All pathways for excision yield 3’OH DNA breakage by transposase is catalyzed in trans Synapsis of the transposon ends is essential for excision RAG and VDJ recombination Nicking to generate 3’ -OH at junction with Nicking to generate V and D segments 3’ -OH in flanking DNA. Attack to generate Hairpin in V and D segments, liberating the Attack to generate ds intersegmental Hairpin in flanking spacer. DNA liberating the transposon. Spacer is functionally a TP Replicative nick and paste transposition cut and paste simple insertion “co-integrate” nick and paste Mu, Tn3, gd Replicative nick and paste transposition (cont.) Co-integrate resolution by conservative site-specific recombination Resolvase catalyzes recombination between res sites LTR elements LTR retrotransposons – intracellular propagation Retroviruses – intercellular propagation LTR Elements (cont.) The transposition of retrotransposons requires an intermediate RNA. LTR and non-LTR elements use different strategies to maintain genome integrity from a derived transcript. transcription The transcript is not a full copy of the viral genome, but contains all information in partial copies of the 5’ and 3’ LTRs Synthesis of long terminal repeat (LTR) element DNA from element RNAs. The complicated conversion of the retroviral RNA to cDNA regenerates intact LTRs in the cDNA to preserve a complete and functional retroviral genome. Integration of cDNA intermediates of LTR elements Integrase catalyzes cut and paste insertion Chemically identical to target joining of DNA-only elements Structural similarity of DNA-only transposases and retroviral integrases Catalytic cores contain spatially clustered DDE or DDD residues, which bind Mg++, essential for transposition Non-LTR retrotransposons LINE (long interspersed element) – autonomous About 6 kb; three LINE families in human genome – only L1 is active LINE elements responsible for most reverse transcription of the genome (retrotransposition of SINEs, generation of processed pseudogenes). SINE (short interspersed element) – non-autonomous About 102 bp; dependent on LINE-encoded proteins for transposition Transposition of non-LTR elements Non-LTR retrotransposons (cont.) Group II mobile introns Bacteria, mitochondria, chloroplasts Catalytic self-splicing RNAs May represent progenitors of nuclear spliceosomal introns of eukaryotic genes Conservative site-specific recombination (CSSR) Recombination supported by short (10’s of bp) DNA sites Catalysis by site-specific recombinases No loss or addition of nucleotides No involvement of DNA polymerases Recombination between inverted repeats (Recombination requires homology) C D C F B E B E F D A A REPEATS INVERTED WITH RESPECT TO EACH OTHER GENERATE AN INVERSION Recombination between direct repeats A B C D C D x F B E E F A Direct repeats (head C D to tail with respect to A F each other) generate an excision/deletion B E The l integrase family of site-specific recombinases “Tyrosine recombinases” - over 100 known l integrase - integration and excision of bacteriophage l Cre recombinase - bacteriophage P1 Flp recombinase - S. cerevisiae Resolution of P1 Replicative Intermediates by Cre loxP Cre loxP A 34-bp loxP site supports recombination by Cre The catalyzed reaction is effectively homologous recombination ATAA… …TTAT ATAA… …TTAT CRE ATAA… …TTAT ATAA… …TTAT Synapsed substrates for Cre-mediated recombination A model for Cre-mediated recombination by means of a Holliday junction intermediate Transposase mediated DNA fragmentation Cut and Paste transposases insert a Tn end at random into DNA, ligating the 3’ end of the transposon to the target DNA insertion site. ATAC Seq Chromatin accessibility for Tn5 mediated oligo insertion ATAC Seq Chromatin accessibility for Tn5 mediated oligo insertion (cont.) Nucleosome mapping Center of fragments of decreasing length converge on the center of a nucleosome 120 200 Transposon mutagenesis Selection Screen Parallel Screen Transposon mutagenesis Transposon mutagenesis Selection Screen Parallel Screen Transposon mutagenesis Broad insertion of transposon Capture of insertion sites as library Sequence Comparison of insertion library before and after growth yields essential gene map. Transposon mutagenesis Essential Genes in Candida glabrata depth of sequencing gene structure (3’insertions) Gale and Cunningham, 2020 Transposon mutagenesis Broad insertion of transposon Capture of insertion sites as library Sequence Comparison of insertion library before and after growth yields essential gene map. Transposon mutagenesis Same library Two growth conditions +/- Flc Below line - sensitive Above line – resistant TCA cycle enzymes Transposon mutagenesis In mice – experiments can yield information relative to complex in vivo environment Cre-activated transposon mutagenesis for cancer gene discovery T2-onc element Engineered oncogenic transposable element derived from Sleeping Beauty transposon Rosa26-loxP-stop-loxP- Sleeping Beauty SB11 transposase under conditional control by Cre Tissue-specific Cre transgene under promoter-Cre control of a tissue- specific promoter Cre-activated transposon mutagenesis Cre-activated transposon mutagenesis for cancer gene discovery (cont.)