3. DNA metabolism: DNA transposition Flashcards
What is the approx. proportion of the human genome that is composed of transposable elements?
Approx. 45%
Who and when discovered transposition?
Barbara McClintock, 1953
What is the main mechanistic difference between DNA and RNA transposons?
DNA transposons employ a “cut-and-paste” mechanism where no replication is involved and only 1 copy of the transposon is made. RNA transposons employ a “copy-and-paste” mechanism which relies on reverse transcription, creating 2 copies of the transposon.
Name 3 reasons for studying transposition
1) Relates to retroviral integration mechanistically and structurally
2) Can be used to study evolution of new cell functions, e.g. V(D)J recombination, CRISPR
3) Have biotechnology adaptations, e.g. CASTs
What is the mechanism and function of DD(E)D superfamily proteins?
Transfer phosphoryl group to rearrange DNA
Name 5 examples of protein families that belong to the DD(E)D superfamily
1) Retroviral integrases
2) DNA transposases
3) Recombinases
4) Resolvases
5) RNAse H
What is the common mechanistic feature of DD(E)D superfamily proteins?
RNAse H like catalytic domain (arginine fingers)
What is the common primary structural feature of DD(E)D superfamily proteins?
Active site triad: D-(90)-D-(34/35/36)-E/D
Name 2 prokaryotic and 1 eukaryotic example of DNA transposons
Prokaryotic: Tn5 and Tn7
Eukaryotic: Mos1
Describe the 4 structural elements of Tn5 transposon
Outside end repeat (19 bp)
Insertion sequence
Inner end repeat
Antibiotic resistance genes x3
What 2 proteins does the Tn5 insertion sequence encode?
The Tn5 transposase and its inhibitor (-1 exon from the transposase)
What does each transposase monomer interact with in cis and trans manner?
Cis - with the DNA strand it recognises
Trans - with the DNA strand it cleaves
How does Tn5 relieve the energetical cost of severe DNA distortion?
By rotating the backbone and flipping out the T2 base, and stacking it with a tryptophan ring
Describe the main steps of Tn5-catalysed DNA cleavage
1) Nucleophilic attack by water of the top strand, generating a 3’-OH
2) Nucleophilic attack by the 3’-OH of the bottom strand, generating a hairpin
3) Nucleophilic attack by water of the hairpin, generating a dsDNA break
Describe the main steps of Tn5-catalysed DNA integration
1) Both 3’-OH ends of the DNA perform a nucleophilic attack on the target sequence with a 9bp stagger
2) DNA repair machinery fills in the 9bp gap, duplicating the target site
Describe the structural elements of the Mos1 transposon (DNA)
Two inverted repeat sequences (28 bp)
Transposase coding sequence (1.3 kb)
What is the recognition sequence of Mos1 transposase?
TA
Name the three protein/DNA complexes that form during Mos1 mediated transposition
1) Paired end (synaptic) complex
2) Post-cleavage complex
3) Strand transfer complex
Why is Mos1 able to perform a dsDNA break without forming a hairpin?
Because cleavage of both strands is performed by a nucleophilic attack by water (hydrolysis)
What base analogue can be used to prove that Mos1 transposition is independent of 3’-OH cleavage?
2,3-dideoxyadenine (ddA)
What structural feature allows Mos1 to cleave two DNA strands of opposite polarity?
Chelation of two divalent metal ions that can switch roles depending on the polarity of bound DNA
What are the roles of the two metal ions in Mos1 transposase?
1) Stabilise the substrate
2) Orient the water molecule for the nucleophilic attack
Describe the structural elements and functions of the Mos1 transposase (protein)
2x helix-turn-helix motifs (sequence-specific recognition)
Catalytic domain containing an RNAse-H-like motif (3 arginine fingers)
Divalent metal ions (1x Mg2+ or 2x Mn2+)
Which key binding partner is different between Mos1/DNA complex during cleavage and Mos1/DNA complex during integration?
During DNA cleavage, flanking donor DNA is bound; during integration, target site DNA is bound