Genetic Recombination

Question 1

What are the 2 types of genetic recombination in bacteria?

Answer 1

• general
• site-specific

Question 2

What does general recombination require?

Answer 2

long (>50bp) sequence homology and RecA

Question 3

What does site-specific recombination require?

Answer 3

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

Question 4

Where does genetic exchange take place?

Answer 4

between 2 pieces of homologous DNA sequences

Question 5

What is the heteroduplex DNA?

Answer 5

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

Question 6

What may recombination result in?

Answer 6

insertion, gene amplification and deletions

Question 7

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

Answer 7

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

Question 8

What is branch migration?

Answer 8

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

Question 9

What does resolution of the Holliday junction produce?

Answer 9

splices or patches

Question 10

What does splice recombinant DNA result from?

Answer 10

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

Question 11

What does patch recombinant DNA result from?

Answer 11

a Holliday junction being resolved by cutting the exchanged strands

Question 12

What is RecBCD and what does it do?

Answer 12

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

Question 13

What are the 3 components of RecBCD?

Answer 13

• RecB = helicase
• RecC = chi site recognition
• RecD = helicase

Question 14

What are chi sites?

Answer 14

crossover hotspot instigator sites that are hotspots for general recombination

Question 15

What is the Ruv complex made up of?

Answer 15

• RuvA
• RuvB
• RuvC
• DNA ligase

Question 16

What is RuvA?

Answer 16

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

Question 17

What is RuvB?

Answer 17

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

Question 18

What is RuvC?

Answer 18

a nuclease that resolves Holliday structures (resolvase)

Question 19

What happens to mismatched DNA in a heteroduplex?

Answer 19

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

Question 20

What are the 3 major classes of transposable elements?

Answer 20

• DNA-only transposons
• retroviral-like transposons
• nonretroviral retrotransposons

Question 21

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

Answer 21

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

Question 22

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

Answer 22

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

Question 23

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

Answer 23

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

Question 24

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

Answer 24

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

Question 25

What is a transposon?

Answer 25

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

Question 26

What are DNA-only transposons responsible for?

Answer 26

the spread of antibiotic resistance in bacterial strains

Question 27

What happens to DNA-only transposons?

Answer 27

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

Question 28

How does transposase function?

Answer 28

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

Question 29

How are DNA-only transposons recognised?

Answer 29

by the inverted repeat DNA sequences present at their ends

Question 30

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

Answer 30

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

Question 31

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

Answer 31

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

Question 32

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

Answer 32

- bacteriophage Mu - retroviruses

Question 33

Describe movement of retroviral-like transposons

Answer 33

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

Question 34

Where is the retrovirus genome packaged?

Answer 34

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

Question 35

What are nonretroviral transposons?

Answer 35

descendants of retroviral DNA that occur as repetitive DNA sequences

Question 36

What happens to circular phage lambda DNA?

Answer 36

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

Question 37

What is attP and attB respectively?

Answer 37

- attP = attachment site on the phage genome - attB = attachment site on the bacterial genome

Question 38

What is cre and what does it do?

Answer 38

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

Question 39

What can site-specific recombination be used for?

Answer 39

to regulate gene expression

Question 40

Why is the recombination enzyme inducible?

Answer 40

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

Question 41

What does tissue-specific gene regulation require?

Answer 41

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

Question 42

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

Answer 42

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