Transposition and transposable elements. Transposable elements “mobile genetic elements” comprise 45% of human chromosomal DNA “middle repetitive DNA”

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1 Transposition and transposable elements

2 Transposable elements “mobile genetic elements” comprise 45% of human chromosomal DNA “middle repetitive DNA” contribute to spontaneous mutation, genetic rearrangements, horizontal transfer of genetic material aid speciation and genomic change (in bacteria transposons are often associated with antibiotic resistance genes) cells must depress transposition to insure genetic stability

3 Types of transposable elements DNA vs. RNA viral vs. nonviral replicative mechanism vs. excision mechanism

4 transposon

5 insertion mutation

6 Discovery of transposons Barbara McClintock 1950’s Ac Ds system in maize influencing kernel color unstable elements changing map position promote chromosomal breaks Rediscovery of bacterial insertion sequences source of polar mutations discrete change in physical length of DNA inverted repeat ends: form “lollipops” in EM after denaturation/reannealing

7 Composite bacterial transposons repeated ends, usually inverted, sometimes direct repeated ends themselves are IS elements and can independently transpose ends mobilize all intervening DNA often antibiotic resistance genes (examples Tn3 (ampicillin), Tn5 (kanamycin), Tn10 (tetracycline) often reside on plasmids

8 Basic minimal insertion sequence structure ends: genetically required, in cis tnp (transposase): genetically required, trans-acting tnp ORF Element 1Element 2Transposition? wt 1 and 2 ends- neither trp-tnp-neither ends-wtonly 2 tnp-wt1 and 2 tnp-ends-only 1

9 Structure of Tn3 4957 bp ampcillin-resistance bla transposase tnpA “repressor” tnpR Tn3 resolvase 3 trans-acting genes: 2 cis-acting sites: 38 bp inverted repeat ends 120 bp “IRS” or res internal resolution site

10 tnpR and res mutations cause accumulation of “co-integrate structure” direct repeat of Tn cointegrate

11 2 types of DNA tranposons excisive mechanism examples: Tn5, Tn10, P elements replicative mechanism examples: Tn3, bacteriophage Mu

12 Replicative transposons orignal cut of transposon is only nick and only one strand is initially ligated element replicates through itself produces as intermediate a “co-integrate” structure co-integrate is resolved by resolvase (as TnpR of Tn3) and at specific site (as res of Tn3)

13 Excisive transposons cut-and-paste mechanism cut themselves out of original site, producing double strand break cut target site and ligate to element ends, thereby inserting at new site original site break repaired usually with sister chromosome, restoring transposon at original site sometimes end healed without transposon, can also be associated with deletion at excision site

14 Source of target site duplication “TSD” GAC CTG GAC Staggered cleavage of target CTG GAC Ligation of transposon DNA GAC CTG Repair replication generates short direct repeats

15 “degenerate” transposons many naturally occurring transposable elements have suffered mutation and are no longer active some of these may have cis-acting end mutations and cannot be mobilized others may have intact ends but no transposase: these can be mobilized by a element that is tnp+ (“autonomous” element) Ac Ds system is an example of latter: Ac (activator) can mobilize Ds (dissociator) MITEs (minature inverted repeat transposable elements) are nonautonomous DNA elements SINEs are retrotransposon version (LINEs)

16 Comparison of transposition reactions Direct transesterification reactions DDE motif transposase (integrase)

17 Comparison of tranposase structural organization

18 Mechanism of transposases and retroviral integrases

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20 Classification of retroelements Have obligate RNA intermediate, use reverse transcriptase (RT, RNA-dependent DNA polymerase) LTR-retroelements: long terminal repeats Ty1/copia, Ty3/gypsy, retroviruses Non-LTR-retroelements “retroposons” LINES

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22 Characteristics of LTR retroelements Long terminal repeats: required for replication cycle Genes: gag, pol, (viruses also have env) Pol is polyprotein which gives rise to RT (reverse transcriptase), IN (integrase) RH (RNase H), PR (protease) Forms VLPs virus-like particles Integrase is functionally and structurally similar to transposase of DNA transposons, DDE motif Integration gives characteristic TSD

23 LTR element replication tRNA primer Multiple template “jumps”

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25 Characteristics of non-LTR retroelements 2 ORFs, orf1, orf2 Variable TSD ORF2 gives rise to EN, endonuclease, (similar to APE) and RT Uses target primed reverse transcription TPRT Can transduce 3’ downstream non-element segments

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27 Target-primed reverse transcription Explains: Insertions are often 5’ truncated Transduction of 3’ markers

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29 Human L1 (LINE-1) retroelement 15% of human DNA 520,000 copies, only 3-5,000 are full-length Associated with human disease loci Transpose specifically in germ line

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