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Genes, Genomes and Biomedical Implications > Genetic Recombination > Flashcards

Genetic Recombination Flashcards

1
Q

What are the 2 types of genetic recombination in bacteria?

A
  • general
  • site-specific
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2
Q

What does general recombination require?

A

long (>50bp) sequence homology and RecA

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3
Q

What does site-specific recombination require?

A
  • very short (<5bp) sequence homology
  • special site recognition
  • specialised proteins
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4
Q

Where does genetic exchange take place?

A

between 2 pieces of homologous DNA sequences

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5
Q

What is the heteroduplex DNA?

A

hybrid DNA from the different parental duplex molecules formed at the site of crossover

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6
Q

What may recombination result in?

A

insertion, gene amplification and deletions

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7
Q

What are the 5 steps of the double strand break model?

A
  1. initial cleavage event to produce protruding ss 3’ tails
  2. ssDNA recognised by RecA which initiates homology search in the other chromosome
  3. ATP-dependent strand exchange followed by DNA synthesis and ligation
  4. branch migration of Holliday junctions
  5. resolution by strand cutting
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8
Q

What is branch migration?

A

process by which the Holliday junction moves along a DNA molecule, allowing for the extension or shortening of the region of heteroduplex DNA formed during recombination

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9
Q

What does resolution of the Holliday junction produce?

A

splices or patches

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10
Q

What does splice recombinant DNA result from?

A

a Holliday junction being resolved by cutting the non-exchanged strands

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11
Q

What does patch recombinant DNA result from?

A

a Holliday junction being resolved by cutting the exchanged strands

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12
Q

What is RecBCD and what does it do?

A

a helicase-nuclease complex that moves along the DNA, separating the strands and generating ssDNA at chi sites

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13
Q

What are the 3 components of RecBCD?

A
  • RecB = helicase
  • RecC = chi site recognition
  • RecD = helicase
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14
Q

What are chi sites?

A

crossover hotspot instigator sites that are hotspots for general recombination

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15
Q

What is the Ruv complex made up of?

A
  • RuvA
  • RuvB
  • RuvC
  • DNA ligase
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16
Q

What is RuvA?

A

a protein that binds to RuvB and Holliday junctions as a tetramer to connect all 4 strands

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17
Q

What is RuvB?

A

a hexamer helicase that catalyses branch migration by binding as a ring around DNA

18
Q

What is RuvC?

A

a nuclease that resolves Holliday structures (resolvase)

19
Q

What happens to mismatched DNA in a heteroduplex?

A

it is recognised and removed by DNA repair enzymes and replaced with a copy of the complimentary strand

20
Q

What are the 3 major classes of transposable elements?

A
  • DNA-only transposons
  • retroviral-like transposons
  • nonretroviral retrotransposons
21
Q

What is the specialised enzyme required for movement of DNA-only transposons?

A

transposase for moving DNA either by cut and paste or replicative pathways

22
Q

What are the specialised enzymes required for movement of retroviral-like transposons?

A

reverse transcriptase and integrase for moving via an RNA intermediate produced in the LTR

23
Q

What are the specialised enzymes required for movement of nonretroviral retrotransposons?

A

reverse transcriptase and endonuclease for moving via an RNA intermediate that is often produced from a neighbouring promoter

24
Q

What can viruses related to DNA-only and retroviral-like transposons do?

A

move in and out of the host cell chromosomes by transpositional mechanisms

25
Q

What is a transposon?

A

a ‘jumping’ stretch of DNA that can jump from one chromosome to another

26
Q

What are DNA-only transposons responsible for?

A

the spread of antibiotic resistance in bacterial strains

27
Q

What happens to DNA-only transposons?

A

they are excised from one spot on a genome and inserted into another

28
Q

How does transposase function?

A

as a dimer; each monomer recognises the same specific DNA sequence at the ends of the transposon

29
Q

How are DNA-only transposons recognised?

A

by the inverted repeat DNA sequences present at their ends

30
Q

When does the cut and paste movement of a DNA-only transposon from one chromosomal site to another begin?

A

when the transposase brings the two inverted DNA sequences together, forming a DNA loop

31
Q

What happens following the transposition reaction of DNA-only transposons?

A

the ss gaps created by the staggered breaks are repaired by DNAP and ligase and as a result the insertion site is marked by a short direct repeat of the target DNA sequence

32
Q

Give examples of viruses that use transpositional site-specific recombination to move into host chromosomes

A
  • bacteriophage Mu
  • retroviruses
33
Q

Describe movement of retroviral-like transposons

A

they move via an RNA intermediate and once the reverse transcriptase has produced a dsDNA, the integrase can recognise a specific sequence near its 2 ends and then insert the viral DNA into the chromosome using a cut and paste mechanism

34
Q

Where is the retrovirus genome packaged?

A

inside capsid proteins which are surrounded by a lipid-based envelope that contains virus-encoded envelope proteins

35
Q

What are nonretroviral transposons?

A

descendants of retroviral DNA that occur as repetitive DNA sequences

36
Q

What happens to circular phage lambda DNA?

A

it is converted to an integrated prophage by a reciprocal recombination between attP and attB

37
Q

What is attP and attB respectively?

A
  • attP = attachment site on the phage genome
  • attB = attachment site on the bacterial genome
38
Q

What is cre and what does it do?

A

a bacteriophage P1 integrase that catalyses site-specific recombination between loxP sites

39
Q

What can site-specific recombination be used for?

A

to regulate gene expression

40
Q

Why is the recombination enzyme inducible?

A

it is encoded by a second DNA molecule that has been engineered to ensure that the enzyme is made only when the animal is treated with a special small molecule, or its temperature is raised

41
Q

What does tissue-specific gene regulation require?

A

the insertion of two specially engineered DNA molecules into the animal’s germ line

42
Q

What are the 2 DNA molecules involved in tissue-specific gene regulation?

A
  • one contains the gene for a recombinase
  • one contains the gene of interest flanked by recognition sites (e.g. loxP) for the recombinase

Genes, Genomes and Biomedical Implications (15 decks)

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