3. DNA metabolism: DNA transposition

Question 1

What is the approx. proportion of the human genome that is composed of transposable elements?

Answer 1

Approx. 45%

Question 2

Who and when discovered transposition?

Answer 2

Barbara McClintock, 1953

Question 3

What is the main mechanistic difference between DNA and RNA transposons?

Answer 3

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.

Question 4

Name 3 reasons for studying transposition

Answer 4

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

Question 5

What is the mechanism and function of DD(E)D superfamily proteins?

Answer 5

Transfer phosphoryl group to rearrange DNA

Question 6

Name 5 examples of protein families that belong to the DD(E)D superfamily

Answer 6

1) Retroviral integrases
2) DNA transposases
3) Recombinases
4) Resolvases
5) RNAse H

Question 7

What is the common mechanistic feature of DD(E)D superfamily proteins?

Answer 7

RNAse H like catalytic domain (arginine fingers)

Question 8

What is the common primary structural feature of DD(E)D superfamily proteins?

Answer 8

Active site triad: D-(90)-D-(34/35/36)-E/D

Question 9

Name 2 prokaryotic and 1 eukaryotic example of DNA transposons

Answer 9

Prokaryotic: Tn5 and Tn7
Eukaryotic: Mos1

Question 10

Describe the 4 structural elements of Tn5 transposon

Answer 10

Outside end repeat (19 bp)
Insertion sequence
Inner end repeat
Antibiotic resistance genes x3

Question 11

What 2 proteins does the Tn5 insertion sequence encode?

Answer 11

The Tn5 transposase and its inhibitor (-1 exon from the transposase)

Question 12

What does each transposase monomer interact with in cis and trans manner?

Answer 12

Cis - with the DNA strand it recognises

Trans - with the DNA strand it cleaves

Question 13

How does Tn5 relieve the energetical cost of severe DNA distortion?

Answer 13

By rotating the backbone and flipping out the T2 base, and stacking it with a tryptophan ring

Question 14

Describe the main steps of Tn5-catalysed DNA cleavage

Answer 14

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

Question 15

Describe the main steps of Tn5-catalysed DNA integration

Answer 15

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

Question 16

Describe the structural elements of the Mos1 transposon (DNA)

Answer 16

Two inverted repeat sequences (28 bp)

Transposase coding sequence (1.3 kb)

Question 17

What is the recognition sequence of Mos1 transposase?

Answer 17

TA

Question 18

Name the three protein/DNA complexes that form during Mos1 mediated transposition

Answer 18

1) Paired end (synaptic) complex
2) Post-cleavage complex
3) Strand transfer complex

Question 19

Why is Mos1 able to perform a dsDNA break without forming a hairpin?

Answer 19

Because cleavage of both strands is performed by a nucleophilic attack by water (hydrolysis)

Question 20

What base analogue can be used to prove that Mos1 transposition is independent of 3’-OH cleavage?

Answer 20

2,3-dideoxyadenine (ddA)

Question 21

What structural feature allows Mos1 to cleave two DNA strands of opposite polarity?

Answer 21

Chelation of two divalent metal ions that can switch roles depending on the polarity of bound DNA

Question 22

What are the roles of the two metal ions in Mos1 transposase?

Answer 22

1) Stabilise the substrate

2) Orient the water molecule for the nucleophilic attack

Question 23

Describe the structural elements and functions of the Mos1 transposase (protein)

Answer 23

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+)

Question 24

Which key binding partner is different between Mos1/DNA complex during cleavage and Mos1/DNA complex during integration?

Answer 24

During DNA cleavage, flanking donor DNA is bound; during integration, target site DNA is bound

Question 25

Where does the Mos1 transposase cleave each strand of the transposon DNA and in which order?

Answer 25

First, bottom strand is cleaved 3bp into the inverted repeat sequence, then top strand is cleaved exactly at the transposon/flanking sequence junction

Question 26

During Mos1-mediated transposition, what performs the nucleophilic attack of the target DNA site?

Answer 26

3’-OH of the transposon DNA

Question 27

What are the 3 methods by which Mos1 integration at TA sequence have been studied?

Answer 27

1) X-ray crystallography - structural studies
2) Fluorescence - biophysical and dynamics studies
3) Mutagenesis - biochemical studies

Question 28

What feature has been added to the target DNA sequence in order to enable crystallisation of the Mos1/target DNA complex?

Answer 28

a CCGG end which creates complementary sticky ends for the complexes

Question 29

How does Mos1 relieve the energetical cost of severe DNA distortion?

Answer 29

By flipping out A1 and filling the space with phenylalanine

Question 30

What method and what base variant can be used to study DNA base-flipping in solution?

Answer 30

Fluorescence studies of DNA containing 2-aminopurine which fluoresces when in a stacked, paired or flipped environment

Question 31

Describe the functional elements of Tn7 transposon (DNA)

Answer 31

From 3’ to 5’: TnsA and TnsB, encode a transposase (heterotetrameric)
TnsC, regulates activity and communicates between TnsA/B and TnsD/E
TnsD and TnsE, alternative proteins that select the target site

Question 32

Name two differences between Tn7 transposition pathways when TnsD or TnsE are involved in target site recognition

Answer 32

1) Tn7 inserts into bacterial chromosome with TnsD, into plasmid with TnsE
2) TnsD transposition maximises vertical transfer, TnsE is involved in horizontal transfer

Question 33

Which of the TnsD/E alternative proteins causes no harm to host?

Answer 33

TnsD

Question 34

Which of the TnsD/E alternative proteins is most similar to DNA replication and why?

Answer 34

TnsE as it interacts with the b-clamp

Question 35

What genes replace tnsD/E in Vibrio cholerae Tn7 transposon?

Answer 35

tniQ and cas6, cas7, cas8

Question 36

What is the function of cas6, cas7, cas8 genes in Vibrio cholerae Tn7 transposon?

Answer 36

Binding of guide RNA and guiding the complex to a specific site in the genome

Question 37

What is TniQ?

Answer 37

A variant of TnsD/TnsE alternative proteins

Question 38

What is the role of TniQ in the V.cholerae Tn7 transposon?

Answer 38

Forms a complex with caspase proteins and recruits TnsC and TnsA/B

Question 39

What is the key difference between caspases used in CRISPR and in CAST?

Answer 39

CAST caspases have no DNA cleavage ability, while CRISPR caspases do

Question 40

What is the main mechanistic difference between CRISPR and CAST, why it arises and how is it advantageous?

Answer 40

Due to dsDNA break induced by Cas9 caspase, integration of new DNA is via homologous recombination which is inefficient. In CAST, DNA integration is performed by the TnsA/B proteins that form part of the Tn7 transposon, which only involves 2x ssDNA breaks that are more efficiently integrated