Lecture 8 - Site Specific Recombination And Transposition

Front 1

CSSR = recombination between 2 _____________.
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Back 1

defined sites

Front 2

CSSR can be used to control ______ expression.

Back 2

gene

DNA inversion can allow an alternative gene to be expressed.

Front 3

Transposition is recombination between....

Back 3

specific sequences and non-specific DNA sites

Front 4

____________________ is the major source of spontaneous mutation

Back 4

Transposition

Front 5

Nearly half the human genome is ______________ derived sequences

Back 5

transposon

Front 6

What are the three types of CSSR

Back 6

insertion, deletion, inversion

Front 7

Which types of CSSR are reversible?

Back 7

Insertion and deletion

Front 8

the recombinase recognition sequences is ______________ while the crossover region within it is ___________________.

Back 8

symmetric, assymetric

Front 9

The crossover region is where DNA ___________ and _________ occur.

Back 9

cleavage, rejoining

Front 10

How many subunits of recombinase bind?

Back 10

Four

Front 11

What are the two types of recombinases?

Back 11

serine and tyrosine

Front 12

Both types use a __________ protein-DNA intermediate.

Back 12

covalent

Front 13

No ____________, such as that released by ______________, is needed for DNA cleavage and joining by these proteins.

Back 13

external energy, ATP hydrolysis

Front 14

The covalent protein–DNA intermediate _______ the energy of the cleaved phosphodiester bond within the protein – DNA linkage.

Back 14

conserves (hence the conservative in CSSR refers to energy; No ATP needed)
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Front 15

The protein-DNA linkage intermediate is called....

Back 15

the synaptic complex

Front 16

Mechanism of serine recombinase:

makes a ____________ stranded break that is staggered by __ bases.

Back 16

double, 2

Front 17

All four strands are __________ before strand exchange.

Back 17

cleaved

Front 18

5' phospho-serine is formed and free ___________ is formed.'

SEGMENTS THEN RECOMBINE

Back 18

3' OH

Front 19

Mechanism of tyrosine recombinase:

Break and rejoin ______ DNA strand at a time.

Back 19

ONE

Front 20

___’- phospho-tyrosine and free ____’-OH formed.
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Back 20

3', 5'

OPPOSITE OF SERINE RECOMBINASE

Front 21

in tyrosine recombinase, a ______________ junction is formed

Back 21

holliday

Front 22

Phage P1 Cre recombinase
is a ___________ recombinase

Back 22

tyrosine

Front 23

The recombination sites on the DNA, where Cre acts, are called _____ sites

Back 23

lox

Front 24

Homologous recombination during
DNA replication of circular chromosomes
can generate circular ___________.

Back 24

multimers

Front 25

_______ is needed to resolve these multimers
back into the ________.

These recombinases are called _________.

Back 25

CSSR, monomers, resolvases

Front 26

_______________________ (resolvase)
is an example from bacteria

Back 26

XER tyrosine recombinase

Front 27

Three classes of transposons

Back 27

DNA transposons,

virus-like retrotransposons/retroviruses (These
elements are also called long terminal repeat (LTR) retrotransposons.) ,

poly-A retrotransposons (non-viral retrotransposons)

Front 28

DNA transposons carry a gene encoding their own ____________. They may carry a few additional genes, sometimes encoding proteins that regulate transposition or provide a function useful to the element or its host cell.

Back 28

transposase

Front 29

The recombination sites are at the two ends of the DNA transposon element and are organized as _________________.
These terminal inverted repeats vary in length from 􏰇25 bp to a few hundred base pairs, are not exact sequence repeats, and carry the recombinase recognition sequences.

Back 29

inverted-repeat sequences

Front 30

The 2 elements/sites in DNA transposons

Back 30

terminal inverted repeats, transposase.

Front 31

Virus-like retrotransposons and retroviruses also carry _____________ sequences that are the sites of recombinase binding and action (Fig. 12-18b).

The terminal inverted repeats are embedded within longer repeated sequences; these sequences are organized on the two ends of the element as direct repeats and are called _______________.

Virus-like retro- transposons encode two proteins needed for their mobility: __________ (the transposase) and _________________.

Back 31

inverted terminal repeat, long terminal repeats (LTRs)

integrase, reverse transcriptase

Front 32

The poly-A retrotransposons do not have the _____________________ present in the other transposon classes.

Back 32

terminal inverted repeats

Front 33

Instead, the two ends of the element have distinct sequences (Fig. 12-18c). One end is called the _______, whereas the other end has a region called the ________ followed by a stretch of ____ base pairs called the _________________.

Back 33

5' UTR, 3' UTR, A:T, poly-A sequence

Front 34

Poly-A retrotransposons carry two genes, known as ORF1 and ORF2.

____ encodes an RNA-binding protein.

____ encodes a protein with both ______________ activity and an ________________ activity.

Back 34

Orf1, orf2, reverse transcriptase, endonuclease/ Rnase H

Front 35

To initiate recombination, the transposase binds to the terminal inverted repeats at the end of the transposon. Once the transposase recognizes these sequences, it brings the two ends of the transposon DNA together to generate a stable protein – DNA complex. This complex is called the ________________ or __________________.

Back 35

SYNAPTIC COMPLEX, TRANSPOSOSOME

Front 36

After excision of the transposon, the ____ ends of the transposon DNA— the ends first liberated by the transposase—attack the DNA phosphodiester bonds at the site of the new insertion.

leave a
new 3’-OH for polymerase
to add onto for the gap to be
filled. Then ligase seals nick

Back 36

3' OH

Front 37

Three mechanisms for cleaving the nontransferred strand. SLIDE 18

Back 37

transposon Tn7 --> another enzyme other than transposase is used (TnsA)

Tn5 and Tn10 --> 3'OH on transposon created by cleaving of transposons ( Tn5 and Tn10) attacks itself creating a hairpin structure. it is opened by transposase

Hermes --> 3'OH on host dna attack itself and create hairpin

last two mechanism creation of hairpin through self cleaving is called

Front 38

last two mechanism creation of hairpin through self cleaving is called

Back 38

transesterification

Front 39

DNA COPY AND PASTE MECHANISM

Back 39

Transposase nicks DNA to create
the 3’-OH at each end for strand transfer
(same as in cut & paste)

3’-OH on each end cleaves
and binds target DNA

DNA replication machinery then uses
each 3’-OH on target DNA as a primer
and copies the transposon


This leaves a large circular DNA
molecule containing 2 copies of
the transposon

Front 40

Virus-like retrotransposons
use an ______ intermediate

Back 40

RNA

Front 41

Virus uses an ___________ related to DNA transposases.
This cleaves ____ nucleotides from the
___’-end, which is then used for DNA strand transfer.

Back 41

integrase, 2, 3'

Front 42

Viruses need to use a special mechanism
to regenerate the ends of the ____
so they can be recognized by ________________.

Back 42

LTRs, integrase

Front 43

target site primed reverse transcription of poly-A is commonly used in Human transposon ______

Back 43

LINEs (long interspersed nuclear elements)

Front 44

When line DNA is transcribed, the line mRNA travels where?

Back 44

to the cytoplasm

Front 45

In the cytoplasm, what is translated from the line mRNA?

Back 45

ORF1 AND ORF 2 protein ELEMENTS

Front 46

Ocnce translated the ORF1 AND ORF2 proteins do what?

Back 46

attach to the 3' end of the line mRNA

Front 47

ORF1 and ORF2 attached to the line mRNA transcript then travels back into the nucleus and do what?

Back 47

Attach to DNA that is T-rich by the use of its poly-A tail. A DNA:RNA hybrid is formed.

Front 48

ORF 2 has two activities, RT and endonuclease. What happens next to the DNA:RNA hybrid VIA ORF2?

Back 48

?

Front 49

Review slide 21

Back 49

plz

Front 50

20% of human genome is _______ elements

Back 50

LINE

Front 51

LINE elements donate proteins (orf1 and orf2) to replicate ______ sequences (13% of human genome).

Back 51

SINE

Front 52

The Alu sequence is an example of a _______.

Back 52

SINE

Front 53

________________ can be made by LINE proteins mistakingly binding and reverse transcribing normal mRNA.

Back 53

Pseudogenes