Genetic Recombination Flashcards
What are the 2 types of genetic recombination in bacteria?
- general
- site-specific
What does general recombination require?
long (>50bp) sequence homology and RecA
What does site-specific recombination require?
- very short (<5bp) sequence homology
- special site recognition
- specialised proteins
Where does genetic exchange take place?
between 2 pieces of homologous DNA sequences
What is the heteroduplex DNA?
hybrid DNA from the different parental duplex molecules formed at the site of crossover
What may recombination result in?
insertion, gene amplification and deletions
What are the 5 steps of the double strand break model?
- initial cleavage event to produce protruding ss 3’ tails
- ssDNA recognised by RecA which initiates homology search in the other chromosome
- ATP-dependent strand exchange followed by DNA synthesis and ligation
- branch migration of Holliday junctions
- resolution by strand cutting
What is branch migration?
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
What does resolution of the Holliday junction produce?
splices or patches
What does splice recombinant DNA result from?
a Holliday junction being resolved by cutting the non-exchanged strands
What does patch recombinant DNA result from?
a Holliday junction being resolved by cutting the exchanged strands
What is RecBCD and what does it do?
a helicase-nuclease complex that moves along the DNA, separating the strands and generating ssDNA at chi sites
What are the 3 components of RecBCD?
- RecB = helicase
- RecC = chi site recognition
- RecD = helicase
What are chi sites?
crossover hotspot instigator sites that are hotspots for general recombination
What is the Ruv complex made up of?
- RuvA
- RuvB
- RuvC
- DNA ligase
What is RuvA?
a protein that binds to RuvB and Holliday junctions as a tetramer to connect all 4 strands
What is RuvB?
a hexamer helicase that catalyses branch migration by binding as a ring around DNA
What is RuvC?
a nuclease that resolves Holliday structures (resolvase)
What happens to mismatched DNA in a heteroduplex?
it is recognised and removed by DNA repair enzymes and replaced with a copy of the complimentary strand
What are the 3 major classes of transposable elements?
- DNA-only transposons
- retroviral-like transposons
- nonretroviral retrotransposons
What is the specialised enzyme required for movement of DNA-only transposons?
transposase for moving DNA either by cut and paste or replicative pathways
What are the specialised enzymes required for movement of retroviral-like transposons?
reverse transcriptase and integrase for moving via an RNA intermediate produced in the LTR
What are the specialised enzymes required for movement of nonretroviral retrotransposons?
reverse transcriptase and endonuclease for moving via an RNA intermediate that is often produced from a neighbouring promoter
What can viruses related to DNA-only and retroviral-like transposons do?
move in and out of the host cell chromosomes by transpositional mechanisms
What is a transposon?
a ‘jumping’ stretch of DNA that can jump from one chromosome to another
What are DNA-only transposons responsible for?
the spread of antibiotic resistance in bacterial strains
What happens to DNA-only transposons?
they are excised from one spot on a genome and inserted into another
How does transposase function?
as a dimer; each monomer recognises the same specific DNA sequence at the ends of the transposon
How are DNA-only transposons recognised?
by the inverted repeat DNA sequences present at their ends
When does the cut and paste movement of a DNA-only transposon from one chromosomal site to another begin?
when the transposase brings the two inverted DNA sequences together, forming a DNA loop
What happens following the transposition reaction of DNA-only transposons?
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
Give examples of viruses that use transpositional site-specific recombination to move into host chromosomes
- bacteriophage Mu
- retroviruses
Describe movement of retroviral-like transposons
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
Where is the retrovirus genome packaged?
inside capsid proteins which are surrounded by a lipid-based envelope that contains virus-encoded envelope proteins
What are nonretroviral transposons?
descendants of retroviral DNA that occur as repetitive DNA sequences
What happens to circular phage lambda DNA?
it is converted to an integrated prophage by a reciprocal recombination between attP and attB
What is attP and attB respectively?
- attP = attachment site on the phage genome
- attB = attachment site on the bacterial genome
What is cre and what does it do?
a bacteriophage P1 integrase that catalyses site-specific recombination between loxP sites
What can site-specific recombination be used for?
to regulate gene expression
Why is the recombination enzyme inducible?
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
What does tissue-specific gene regulation require?
the insertion of two specially engineered DNA molecules into the animal’s germ line
What are the 2 DNA molecules involved in tissue-specific gene regulation?
- one contains the gene for a recombinase
- one contains the gene of interest flanked by recognition sites (e.g. loxP) for the recombinase
