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Molecular Biology > Chapter 11 > Flashcards

Chapter 11 Flashcards

1
Q

What are some of the roles of site specific recombination?

A
  1. Virus phage insert their chromosomes via site specific recombination mechanisms,
  2. Site specific recombination is used to alter gene expression, like inversion of DNA segment to allow two alternative genes to be expressed.
  3. widely used to help maintain chromatin structure
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2
Q

What are the two classes of genetic recombination?

A 
Conservative Site-Specific Recombination (cssr) &
Transpositional Recombination (transposition)
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3
Q

What is the major source of spontaneous mutation is most organisms?

A

Transposition

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4
Q

What are proteins that recognize specific sequences where recombination will occur within a DNA molecule? they are present in both cssr & transposition mechanisms.

A

Recombinases

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5
Q

When recombinases bring specific sites together to form a protein-DNA complex “bridging” the DNA sites, what is this called?

A

Synaptic Complex

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6
Q

What is a recombination site in CSSR?

A

segment of DNA that will be moved which carries specific short sequence elements, “recombination sites”, where DNA exchange occurs.

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7
Q

each recombination site is organized as a symmetrical pair of?

A

Recombinase recognition sequences

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8
Q

Recognition sequences are positioned symmetrically and flank a central short asymmetric sequence that is the location of DNA cleavage and joining. What is the sequence called?

A

Crossover region

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9
Q

The orientation of the recombinase recognition sequence and crossover region is an inverted repeat, what results?

A

recombination between a pair of inverted sites will invert the DNA segment between the two sites

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10
Q

The orientation of the recombinase recognition sequence and crossover region is a direct repeat, what results?

A

Recombination between a pair of direct repeats will delete the DNA segment between the two sites

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11
Q

What happens when recombination sites on two different molecules are brought together for DNA exchange?

A

Insertion

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12
Q

What are the two families of conservative site-specific recombinases?

A

Serine Recombinases & Tyrosine Recombinases

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13
Q

Why is it called “conservative” site-specific recombination?

A

The mechanistic feature in which every DNA bond that is broken during the reaction is resealed by the recombinase. -No external energy, such as that released by ATP hydrolysis, is needed for DNA cleavage and joining by these proteins.

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14
Q

DNA topoisomerases, Spo11 and Recombinases all share this mechanistic feature?

A

They are “conservative” in its cleavage mechanism with it’s covalent intermediate.

Conservate means that no external energy is needed for DNA cleavage, and every DNA bond that is broken during the reaction is resealed.

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15
Q

Which recombinase introduces double-strand breaks in DNA and then swap strands to promote recombination?

A

Serine Recombinases

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16
Q

Which recombinase has a structure which suggests rotation to achieve strand exchange?

A

Serine Recombinase, as a large, flat tetramer interface that is the site of rotation - all four DNA strands must be cleaved by the recombinase.

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17
Q

Which recombinase breaks and rejoins one pair of DNA strands at a time?

A

Tyrosine recombinases

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18
Q

Accessory proteins which bind specific DNA sequences and bend the DNA into a specific shape to stimulate recombination?

A

Architectural proteins

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19
Q

Which type of protein ensures DNA segment integration occurs without the reverse reaction of DNA excision?

A

Architectural proteins

-can control the direction of a recombination reaction

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20
Q

What promotes integration and excision of a viral genome into the host-cell chromosome?

A

λ integrase

-when bacteriophage λ infects a host bacterium, a series of regulatory events result either in establishment of the quiescent lysogenic state or in phage multiplication, a process called lytic growth

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21
Q

Try and highlight the differences between Cre-recombination and λ-integration?

A

The mechanisms are the same, as CSSR by Tyrosine, and break/rejoining one DNA strand at a time. λ-integrase requires the use of architectural proteins or accessory proteins, while Cre-recombination does not.

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22
Q

What are the purpose of the architectural proteins (accessory proteins) required for λ-integration?

A

These proteins control the reaction to ensure that DNA integration and DNA excision occur at the right time in the phage life cycle.

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23
Q

What architectural protein is a sequence dependent DNA-binding protein that introduces large bends (>160º) in DNA?

A

Integration Host Factor (HIF)

in λ-integration, integration at attB, attP and λInt all require HIF.

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24
Q

What inverts a segment of DNA allowing expression of alternate genes in Salmonella?

A

Hin Recombinase

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25
What phage encoded protein is required for excessive recombination, and works by binding three sites introducing a >140º bend?
Xis, an architectural binding protein, has the opposite effect as IHF.
26
What is it about salmonella Hin inversion that is able to express one gene and not the other?
When the invertible segment is in the "on" position, there is a functional promoter, that when inverted, is no longer functional and is considered "off". Each conformation expresses a different flagellar gene and is therefore useful when immune systems are fight off a certain one, then they can switch to the other.
27
What is a site-specific DNA-bending protein that makes protein-protein contacts with Hin that are important for recombination?
Factor For Inversion Stimulation (Fis) ~60 bp sequence is an enhancer that stimulates the rate of recombination ~1000 fold. The Fis-enhancer complex activates the catalytic steps of recombination.
28
True or false, Hin can actually assemble and pair the hix recombination sites to form a synaptic complex in the absence of the Fis-enhancer complex?
True, for Fis activation of Hin, the three DNA sites (HixL, hixR, and enhancer) need to come together, forming a 3-way complex which is greatly facilitated by negative DNA supercoiling, that stabilizes the association of the distant DNA sites.
29
What bacterial architectural protein also facilitates assembly of Hin-invertasome complex?
HU -is a close structural homolog of IHF, but it contrast, it binds DNA in a sequence-independent manner.
30
What is the biological rationale for control of Hin inversion by the Fis-enhancer complex?
The principal function is to ensure that recombination occurs only between hix sites that are present on the same DNA molecule. This selectivity ensures that the invertible segment is flipped frequently but also that intermolecular DNA rearrangements, which could disrupt the integrity of the bacterial chromosome, are avoided.
31
In contrast to integration and excision of bacteriophage λ, Hin-catalyzed inversion is not highly regulated, but inversion occurs stochastically. What does this mean?
Hin-catalyzed inversion occurs stochastically such that within a population of cells, there will always be some cells that carry the invertible segment in each orientation.
32
What can convert multimeric circular DNA molecules into monomers?
Recombinases | also "resolvases" right?
33
An intrinsic problem with circular DNA molecules is that they sometimes form dimers and even higher multimeric forms during the process of homologous recombination. What can be done to convert these back into monomers?
By site-specific recombination recombinases called "Resolvases". Although the specific one mentioned in the book, "FtsK", is said to be a "cell division protein" that assists the Xer family recombinases. Site-specific recombination can be used to convert these DNA multimers back into monomers. Many circular DNA molecules carry sequences recognized by resolvases.
34
Form of genetic recombination, Transposition, moves what genetic elements from one DNA site to another?
Transposable elements or transposons
35
What are the recombinases responsible for transposition called?
Transposases, or sometimes Integrases
36
After page 339, need flashcards
after page 339 need flashcards
37
What does CSSR stand for?
Conservative Site Specific Recombination
38
Example of CSSR given in class?
Lambda phage inserting into bacterial chromosome
39
what are the three types of CSSR? | -note the direction-
Insertion - (a-☞-b) + (x-☞-y) = (x-☞-a--b-☞-y) Deletion - (x-☞-a--b-☞-y) = (a-☞-b) + (x-☞-y) Inversion - (x-☞-a--b-☜-y) = (x-☞-b--a-☜-y)
40
What are the general steps in CSSR?
1. Crossover regions line up recombinase recognition sequences 2. Recombinase binds recognition sequences 3. Recombination
41
Describe the covalent intermediate mechanism used by serine and tyrosine recombinases?
(Rec)-(Ser/Tyr)-OH attacks phosphate backbone, 3' O on above nucleotide accepts H, ...imagine this intermediate with (Rec)-(Ser/Tyr)-O-PO3-sugar etc... Then to end it, the 3' hydroxyl attacks the same P displacing recombinase.
42
What is unique about Serine recombinase structure?
Contains a swivel area. It's mechanism is "cleavage––DNA swap partners––rejoining"
43
What is the mechanism of a Tyrosine Recombinase?
Attack to form P-Tyrosine–––Top strand exchange to form "holiday junction"–––Cleavage of bottom strands–––Bottom strand exchange to finish recombination.
44
What is Cre recombinase and what is a general characteristic of it's mechanism?
Cre recombinase is a "Tyrosine Recombinase" which cleaves and joins one strand at a time.
45
What is the difference in the proteins required for Lambda Phage integration and Lambda Phage deletion?
Integration - Int, Hif | Deletion - Int, Hif, Xis
46
What binds to H-sites and introduces large bends in the DNA, which allows the λInt to bind to P sites and be close to the CSSR sites, attP and attB or attL and attR?
Integration Host Factor (IHF)
47
In Salmonella, what are the respective directions of the Hix sites when converting H1 to H2 flagellar antigen?
The hixL and hixR CSSR sites are opposite in orientation. Recombination results in inversion of the segment. Remember that hix's here are "crossover" regions. This makes sense according to the directions of the three types of CSSR's.
48
T/F, Factor for Inversion Stimulation (Fis) binds to enhancer sequence and the Hin recombinase, stimulating the recombination?
True, just Hin will yield Hin-Hix synaptic sequence Hin with Fis will result in an "invertasome"
49
A homologous recombination event to repair a ds break will result in a dimer chromosome, what is the result of subsequent CSSR to the dimer?
CSSR will yield two monomers.
50
Outline CSSR with XerC XerD recombinases acting with Fitsk and without Fitsk?
Without Fitsk, XerC will only be able to swap one strand while XerD is inactive and cannot finish the job. With Fitsk, XerC swaps it's strand, while Fitsk binds and bends the complex to activate XerD to swap it's strand, and recombination can complete.
51
T/F, with no crossovers XerC and XerD can separate the chromosomes from midcell line? What if there are an "odd" number of crossovers?
True, with no crossovers all you need is XerC and XerD to finish the job. When there are crossovers, Fitsk needs to be active after the "division site closure line" in order to activate XerD to recombine & segregate completely into two cells.
52
What are two ways in which genetic transposition can occur?
1. Transposon movement without duplication, from one site to another 2. Transposon movement with duplication, copied from from site and inserted into another.
53
T/F, 1,400 Mb of genome wide repeats are forms of transposable elements?
True
54
Three types of transposons?
DNA Virus Like retrotransposons/retroviruses Poly-A retrotransposons
55
Which type of transposon has terminal inverted repeats and transposase in the center element?
DNA transposons
56
Which type of transposon has integrase and RT in its element?
Virus like retrotransposons/retroviruses
57
Which type of transposon has no terminal repeats but has three A-T nucleotide stacks on the end?
Poly-A retro transposons
58
What are the major types of DNA-mediated transposition?
Bacterial Replicative Transposons Bacterial Cut-And-Paste transposons Eukaryotic Transposons
59
What are the major types of RNA mediated transposons?
Virus-like retrotransposons | Poly-A retro transposons
60
What is a complication of a high Tn10 copy number?
RNA:RNA pairing is frequent which blocks translation of transposase mRNA when there is little Tn10 copy number RNA:RNA pairing is rare and translation of transposase mRNA is efficient.
61
Phage Mu is a lysogenic bacteriophage. It is also a large DNA transposon. During lytic phase, Mu uses multiple rounds of transposition to replicate 100 times. So how does it prevent transposing it's own DNA? Mu uses transposition target immunity to prevent transposing into its own DNA.
Mu uses transposition target immunity to prevent transposing into its own DNA.
62
What element in yeast is similar to a virus like retro transposon?
Yeast Ty elements are viral-like retrotransposons. Ty elements select specific regions to transpose. Ty1 200 bp from RNA pol III promoter Ty3 2 bp from RNA Pol III promoter Ty5 silenced regions
63
What are the most common DNA transposons in eukaryotes
Tc1/Mariner Elements they are Autonomous poly-A Retrotransposons
64
Outline the process of VDJ recombination?
``` [germ-line DNA] ↓ DNA rearrangement during B-cell differentiation [B-cell DNA] ↓ Transcription [Transcribed RNA] ↓ RNA splicing [Spliced mRNA] ↓ Translation **Light Chain Protein** (VJC regions) ```
65
What are the proteins involved in V(D)J recombination during the first step of single strand cleavage?
RAG1, RAG2, HMG protein
66
What intermediate structure is formed during V(D)J cleavage?
Hairpin formation
67
What happens to the Signal Joint formation in V(D)J recombination?
It is discarded
68
After hairpin formation, what is the next step in V(D)J?
Hairpin opening and fusion of coding segments ↓ Further recombination
69
How does recombination always occur in V(D)J?
between a pair of recombination signal sequences in which one partner has the 12-bp "spacer" and the other partner has the 23-bp "spacer" – which are organized as Inverted Repeats flanking the DNA segments that are destined to be joined.
70
What are the functions of RAG1 and RAG2 in V(D)J?
recombinase that is responsible for recognizing and cleaving the recombination signal – function in a manner very similar to that of a transposase by recognizing recombination signal sequences and paring the two sites to form a protein-DNA synaptic complex.
71
What does RAG stand for?
recombination-activating gene
72
Packaged RNA genome and tRNA primer slide? What is it?
????

Molecular Biology (10 decks)

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