Chromosome biology lecture 7

Question 1

Transpositional recombination overview

Answer 1

• Similar to SSR as involves protein DNA + protein-protein interactions
• Only 1 of the recombining partners is specifically recognised by the protein that catalyses transposition

Question 2

3 general mechanisms for genetic mobility

Answer 2

1. SSR
2. Transposition - transposes binds inverted DNA at end of element, each transposes cleaves ends of elements + transfers to new molecule
3. Target-primed reverse transcription - transposable element copied from its DNA by RNA pol + produces RNA copy of element → encodes reverse transcriptase that nicks target DNA + element copied in

Question 3

Transposable elements

Answer 3

• Abundant, 12% of c elegans genome
• Most a silent, some are active
• Specific DNA/RNA elements can move
• Source of spontaneous mutation
• Can lead to gene rearrangements

Question 4

Classification of eukaryotic transposable elements

Answer 4

1. Class I = retrotransposons, transpose via RNA int, use reverse transcriptase, split into LTR, non-LTR or DIRS
2. Class II - DNA transposons

Question 5

LTR transposons

Answer 5

• Inc retroviruses like HIV
• Both use integrate
• Retroviruses can be distinguished from LTR transposons by presence of envelope gene located at 3’

Question 6

Non-LTR transposons

Answer 6

• x have LTR
• Transposes via target-primed reverse transcriptase
• uses endonuclease to nick DNA (restriction-like or AP)
• e.g. = LINES/ SINES

Question 7

DNA transposons

Answer 7

• Divided into autonomous elements + non-autonomous

Question 8

Classification of prokaryotic transposable elements

Answer 8

1. Simple insertion sequences (800-1500bp, inverted terminal repeats flanking gene for transposase)
2. Compound transposons (have 2 insertion sequences which cooperate to transpose own DNA as well as DNA in btw)
3. Complex transposon (terminal inverted repeat flanking range of genes)

Question 9

Transposable element abundance + activity

Answer 9

• Most common = Tc1/Marnier family

- Only around 10 elements are active inc P elements in Drosphilia

Question 10

Mechanism of transposition

Answer 10

• Liberation from donor DNA + insert/joining to target DNA

- Can either paste in or copy w/ reverse transcriptase

Question 11

Transposition types

Answer 11

Conservative vs replicative

Both use endonucleolytic cleavage of phosphodiester bonds, transfer ends into target DNA

Question 12

Conservative (cut + paste) transposition

Answer 12

• Tc1/Marnier family
• Typically these transposes = DBD, CT catalytic domain,
• Asp + Glu coordinate 2 Mg2+
• 2 transposes recognise inverted repeats, bind w/ HTH → ‘single end’ complex
• Cleave 5’ of inverted repeat → ‘paired end complex’
• 3’ end cleaved + release 3’OH termini
• Bind target DNA
• 2 strand cleavages liberate from end of donor DNA
• Fill in w/ DAN pol
• Transposon footprint

Question 13

Coordination of transposition w/ DNA replication

Answer 13

• Conservative = wasteful
• Overcome by coordinate
• Excision of transposon ensures ds break is repaired by HR

Question 14

Replicative transposition

Answer 14

• Only 1 strand cleaved at each transposon end
• Strand transfer of cut 3’ end into target DNA → Shapiro int.
• 2 3’ ends → replication of transposon
• Intermolecular replication transposition → co-integrate structures where donor + target replicon are joined but separated by directly repeated copy of element
• Resolution

Question 15

Life cycle of DNA transposon

Answer 15

• Retrotransposons are transcribed
• Assoc. w/ mRNA
• In retroviruses, envelope gene means mRNA + proteins → infectious particles by bonding off
• In virus-like particles, pol derived protease cleaves protein
• RNA reverse transcribed to comp. DNA

Question 16

Reverse transcription of retroviral DNA

Answer 16

• 3’ end of host tRNA binds 5’ of viral RNA
• Reverse transcriptase makes short segment of RNA
• RNase H exposes 3’ end of - DNA
• tRNA mostly degraded
• Short stretches of RNA persist, primes synthesis of + DNA which proceeds to 5’
• 2nd template switch

Question 17

Integrase integration

Answer 17

• Integrates into cDNA of host

- Generates 2 base recessed 3’ ends in LTR + staggered ends in target DNA

Question 18

Strand cleavage

Answer 18

• Transposition requires assembly of synaptic complex
• Precise cleavage to liberate 3’OH
• Cleavage of compl strand sometimes occurs
• Hairpins hydrolysed → 3’OH
• 3’ ends transferred into target DNA

Question 19

Disadv of transposition

Answer 19

• Insertion near genes can Δ gene expression (can carry gene promoter)
• Indirectly harm by acting as sites for non-allelic HR

Question 20

Transposable elements targeting specific sites

Answer 20

• E.g. in some yeast specifically target promoter regions or genes transcribed by RNA pol III
• E.g. TyI integrase in yeast has LTR that interacts w/ subunit of RNA Pol III

Question 21

Regulation of transposable elements

Answer 21

• small RNAs silence
• KRAB domain (>400 genes), bind transposable elements in a sequence-specific manner + recruit KAP1 → H3 lys 9 methyl transferase recruited → epigenetic silencing
• De-reg = hallmark of cancer

Question 22

Adv of transposition

Answer 22

• Antibiotic resistance
• VDJ recomb = antigen receptor diversification (relies on transposition-like reaction + NHEJ, RSS bound by VDJ recombinase which initiates recombination

Question 23

Transposon as genetic tool

Answer 23

• Typical gene transfer = 2 plasmids

- ↑ efficiency of transposons in catalysing genomic insertion