Genome Integrity 2

Question 1

What are the two major groups of transposable elements?

Answer 1

DNA-only intermediates and RNA intermediates

Question 2

What are the different types of transposable elements that go through RNA intermediates?

Answer 2

LTR elements and non-LTR elements

Question 3

What are the two types of DNA-only transposons?

Answer 3

Cut and paste elements and replicative nick and paste elements

Question 4

Three major bacterial DNA transposon superfamilies?

Answer 4

IS4 (Tn5, Tn10), Mu, Tn3/gamma delta

Question 5

What elements do transposases act on in DNA-only transposons?

Answer 5

IS elements

Question 6

Mechanism: Cut and Paste Transposition

Answer 6

1) transposase bind and pairs both ends of IS elements
2) transposase cuts out entire IS element and exposes 3’ OH at each end of element
3) 3’ OH of IS element can attack target DNA phosphodiester backbone in a sequential fashion
4) Gap repair fills in ssDNA

Question 7

How does transposase catalyze DNA breakage?

Answer 7

Catalyzes DNA breakage in trans. One protomer of transposase binds the DNA, but the other protomer facilitates cleavage of the DNA to expose 3’ OH.

Question 8

Mechanism: Replicative Nick and Paste Transposition

Answer 8

1) MuA transposase makes nick at 3’ ends of Mu
2) 3’ OH of Mu attacks target DNA at staggered positions
3) Mu gets replicated starting from flanking target DNA
4) Mu gets replicated and two copies of Mu link donor and target DNA
5) Resolvase will split 2 plasmids between res sites in TE (homologous recombination)

Question 9

What are the two types of LTR elements?

Answer 9

Retroviruses and retrotransposons

Question 10

What are the differences between the two types of LTR elements?

Answer 10

Retroviruses can bud out. Retrotransposons lack packaging proteins, so they immediately go through cDNA intermediate. In the end, they all integrate back into the genome.

Question 11

How do LTR elements integrate into the genome?

Answer 11

Similar mechanism to DNA-only transposons. Reverse transcription to cDNA creates 3’ OH on each side. These 3’ OH’s facilitate transposon DNA insertion.

Question 12

How are DNA-only transposases and retroviral integrases related?

Answer 12

Have structural similarities in their catalytic site. Spatially clustered DDD or DDE binds Mg2+. This is essential for transposition

Question 13

What are the two types of non-LTR retrotransposons?

Answer 13

LINE (long interspersed elements) and SINE (short interspersed elements)

Question 14

Mechanism: Non-LTR transposition

Answer 14

1) LINE element gets transcribed into LINE RNA (with Poly A tail)
2) LINE endonuclease makes nick at target site (usually AT track)
3) Poly A tail of LINE can pair with nicked target site (T’s)
4) Reverse Transcriptase uses LINE as a template to synthesize DNA
5) SOMETHING nicks the second target site and DNA synthesis is completed using second strand as template
6) have to replace RNA with DNA and fill in single stranded gaps

Question 15

Function: RME Complex

Answer 15

Reverse transcriptase, Maturase, Endonuclease which are encoded by group II mobile introns. Facilitates transposition of these introns.

Question 16

Mechanism: Group II Mobile Intron

Answer 16

1) G2MI is transcribed and RME is translated
2) RME facilitates splicing of intron lariat
3) RME Maturase reverse splices intron RNA into target
4) RME endonuclease makes a nick in DNA
5) RME reverse transcriptase makes cDNA using RNA as template
6) RNA gets degraded and DNA processing inserts intron into target site

Question 17

How is conservative site-specific recombination facilitated?

Answer 17

By λ integrase family of recombinases

Also known as Tyrosine recombinases because it goes through tyrosine intermediate

Question 18

What constitutes a loxP site?

Answer 18

34 base pairs with two 14 bp recombinase binding elements (RBE) and 6 bp crossover region.

Question 19

What does Cre recombinase bind to in the loxP site?

Answer 19

They bind to the RBE sites.

Question 20

How does Cre mediate recombination?

Answer 20

Only one subunit is active at a time

1) One subunit on each duplex nicks DNA and makes 3’ P-tyr with exposed 5’ OH
2) 5’ OH ends are exchanged between DNA duplexes
3) Exchanged 5’ OH attacks 3’ P-Tyr to rejoin DNA
4) Do the same mechanism with non-exchanged strand using other subunit per LoxP active site.