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LSM2232 > DLW05 - Genetic Recombination > Flashcards

DLW05 - Genetic Recombination Flashcards

1
Q

State two differences between general recombination and site-specific recombination.

A
  1. General recombination requires a long sequence homology but site-specific recombination requires very short sequence homology.
  2. General recombination is RecA dependent but site-specific recombination is RecA independent.
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2
Q

General recombination can never occur within the same DNA molecule. TRUE or FALSE?

A

FALSE. General recombination may be intra- or inter- molecular events.

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3
Q

Define the term “heteroduplex DNA”

A

Heteroduplex DNA is hybrid DNA produced from the different parental duplex molecules.

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4
Q

State the four outcomes of recombination

A
  1. Insertion
  2. Deletion
  3. Duplication (Amplification)
  4. Inversion
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5
Q

There are two models of general recombination. Name the correct model.

A

Double strand break model.

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6
Q

Briefly describe the process of general recombination using the double strand break model.

A
  1. The same DNA molecule is nicked on both strands.
  2. The double stranded break undergoes limited degradation by a 5’ to 3’ exonuclease to create protruding single-stranded 3’ tails. (by RecBCD)
  3. Single stranded DNA are recognised by RecA protein which initiates homology search in the other chromosome.
  4. ATP-dependent strand exchange occurs, followed by synthesis and ligation.
  5. Branch migration occurs, increasing the distance between the two Holliday junctions.
  6. Resolution occurs by strand cutting.
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7
Q

State the function of RecA

A

RecA is a protein which initiates homology search during strand invasion.

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8
Q

Define the Holliday junction.

A

The Holliday junction is an intermediate structure in homologous recombination, where two duplexes of DNA are connected by the genetic material exchanged between two of the four strands.

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9
Q

Define the term “branch migration”.

A

Branch migration describes the ability of a DNA strand partially paired with its complement in a duplex to extend its pairing by displacing the resident strand with which it is homologous. This process is ATP-dependent.

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10
Q

Describe what happens when exchange strands are cut during the resolution of a Holliday junction.

A

If exchange strands are cut, the duplex remains largely unchanged. Patch recombination DNA is formed.

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11
Q

Describe what happens when non-exchanged strands are cut during the resolution of a Holliday junction.

A

If the non-exchanged strands are cut, splice recombinant DNA will be formed. Both strands of DNA before the exchange point will come from one chromosome, while the DNA after the exchange point will come from the homologous chromosome.

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12
Q

State the function of RecBCD

A

RecBCD is a helicase-nuclease complex, involved in the initiation of repair of DSBs.

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13
Q

Explain how RecBCD carries out its function.

A

RecBCD is a helicase-nuclease complex. RecBCD uses its helicase activity to unwind double stranded DNA. Its 5’ to 3’ exonuclease and 3’ to 5’ exonuclease then allow it to degrade the DNA molecule. Once RecBCD encounters a chi site, its 3’ to 5’ exonuclease activity is suppressed. This generates a 3’ terminal single stranded end.

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14
Q

What are chi sites?

A

Chi sites (crossover hotspot instigator) are hotspots for general recombination. The chi sites signal the suppression of RecBCD’s 3’ to 5’ exonuclease activity, allowing for the generation of a 3’ terminal single stranded end for strand invasion.

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15
Q

State the family of proteins that are involved in resolving Holliday junctions.

A

Ruv (Ruv A, B and C)

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16
Q

State the function of RuvA.

A

RuvA binds to RuvB and Holliday junctions.

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17
Q

State the function of RuvB.

A

RuvB is a helicase that catalyses branch migration

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18
Q

State the function of RuvC

A

RuvC is a nuclease (resolvase) that resolves Holliday structures.

19
Q

What is meant by non-reciprocal exchange?

A

In recombination, non-reciprocal exchange would mean that the donor strand remains unchanged.

20
Q

Briefly describe what happens for gene conversion to occur.

A

During general recombination, mismatched DNA in a heteroduplex are recognised and removed by DNA repair enzymes and replaced with a copy of the complementary strand.

21
Q

It is possible for gene conversion to not occur even with base mismatches. TRUE or FALSE? Explain why.

A

TRUE. If DNA replication occurs before DNA repair, mismatched bases on both strands will not be repaired. Instead, both daughter copies will now carry their own allele. No gene conversion has occured.

22
Q

State the three mechanisms of site-specific recombination covered.

A
  1. Transposons
  2. Phage integration and excision
  3. Cre-LoxP system
23
Q

State the three major classes of transposable elements.

A
  1. DNA-only transposons
  2. Retroviral-like retrotransposons
  3. Nonretroviral retrotransposons
24
Q

Explain what is meant by “cut-and-paste mechanism”

A

The cut-and-paste mechanism entails the excision of the transposon from one spot of a genome an insertion into another.

25
Q

Transposition occurs very frequently in bacteria due to their rate of replication. TRUE or FALSE?

A

FALSE. Transposition is rare.

26
Q

Describe the structure of a Class I transposon

A

The transposon is flanked by short inverted repeats (SIR). The transposon contains the transposase gene, as well as other beneficial genes (such as antibiotic resistance genes).

27
Q

Suggest how one would identify a Class I transposon.

A

Identifying the short inverted repeats flanking a transposase gene would confirm the presence of a transposon. However, short direct repeats are also indicative of a possibility that a transposon is in the genome sequence.

28
Q

Briefly describe the cut-and-paste mechanism.

A
  1. Transposase monomers are encoded for by the transposase gene within the transposon.
  2. Each individual monomer binds to the two short inverted sequences flanking the transposon.
  3. The monomers dimerise to form the transpososome, and the transposon is excised.
  4. A staggered cut is made on the target site, allowing for the transposon to be integrated.
29
Q

State the enzymes involved in retroviral-like retrotransposition.

A

Reverse transcriptase and integrase (viral transposase)

30
Q

What distinguishes non-retroviral retrotransposons?

A

The presence of a poly-A tail.

31
Q

State the difference between an endonuclease and an exonuclease.

A

An endonuclease cleaves the phosphodiester bond from the interior, while an exonuclease does so from the exterior.

32
Q

Briefly describe non-retroviral retrotransposition

A
  1. RNA synthesis of the transposon occurs first.
  2. The RNA encodes for reverse transcriptase and an endonuclease which form an endonuclease-reverse transcriptase complex.
  3. The complex nicks the target DNA on one strand and proceeds to synthesise the DNA strand using the RNA as a template.
  4. A second DNA strand is formed before integration into the target site.
33
Q

State the enzymes required for phage integration

A

Integrase (Int) and integration host factor (IHF)

34
Q

State the enzymes required for phage excision.

A

Integrase (Int), integration host factor (IHF) and excisionase (Xis).

35
Q

How is attL identified?

A

attL contains the left portion of attP (POB’)

36
Q

State the experimental technique developed based off phage excision and integration.

A

Gateway cloning system

37
Q

attB and attP are directional. TRUE or FALSE?

A

TRUE.

38
Q

What do attB and attP stand for?

A

attB (bacteria); attP (phage)

39
Q

In an experimental setting, how would attB and attP flanks be generated?

A

PCR

40
Q

What is the Cre protein?

A

Cre is a bacteriophage P1 integrase which catalyses site-specific recombination between loxP sites.

41
Q

The Cre-loxP system has only been able to work in vitro. TRUE or FALSE?

A

FALSE. This form of recombination works in vitro and in vivo.

42
Q

Briefly describe how the Cre-loxP system can be used in an experimental setting.

A

The Cre-loxP system can be used for temporal and spatial regulation. It can be used to either induce or silence target genes.

43
Q

Describe how Cre-loxP can be used to induce the expression of a target gene X.

A

The target gene can be placed downstream of the loxP sites. The promoter should be upstream of the loxP sites. When the recombinase Cre is expressed, excision of the marker gene within the loxP sites result in the target gene being immediately downstream of the promoter, switching on the gene.

44
Q

Describe how Cre-loxP can be used to silence the expression of a target gene Y.

A

The target gene can be placed in between the two loxP sites. When the recombinase Cre is expressed, the target gene is deleted and lost as the cells divide.

LSM2232 (9 decks)

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