Genetic Recombination Flashcards
What are the types of genetic recombination in bacteria?
General recombination
Site-specific recombination
What does general recombination require?
Needs RecA
Requires long sequence homology = more than 50 bp
What does site-specific recombination require?
Doesn’t need RecA but needs specialized proteins
Special site recognition
Requires v short sequence homology = less than 5 bp
What is genetic recombination?
Genetic exchange between 2 homologous DNA sequences
Heteroduplex formation at the site of crossover
New recombinant DNA molecules are made
How does recombination come about in the double strand break model?
Limited degradation at double-strand break by 5’ to 3’ exonuclease
Single-stranded 3’ tails
ssDNA recognized by RecA
RecA initiated homology search in the other chromosome
ATP needed for strand exchange occurs
DNA synthesis and ligation
Branch migration of Holliday junctions
Resolution by strand cutting
Define branch migration
Ability of DNA strand partially paired with its complement in duplex = to extend its pairing by displacing resident homologous strand
ATP dependent
Define Holliday junction
Intermediate structure in homologous recombination
Two duplexes of DNA are connected by genetic material exchanged between two of the 2 strands
What are the two types of recombinants produced from resolution of Holliday junctions?
Splice recombinant
Resolution by cutting non-exchanging strands = DNA after exchange point comes from the homologous chromosome
Patch recombinant
Resolution by cutting exchange strands = duplex largely unchanged except for DNA sequence on 1 strand that came from homologous chromosome
What is RecBCD function?
Helicase-nucleaus complex that initiates the repair of double-strand breaks
Recognizes double strand break
What site does RecBCD recognize?
Chi site = hotspot for general recombination
What happens when RecBCD reaches chi site?
Nuclease activity with 3’ end is supressed
Other strand continues to be degraded = produce 3’ single stand
ssDNA coated by RecA for homologous recombination
Describe RuvA
Binds RuvB and Holliday junctions
Tetramer = contacts all 4 strands
22kDa
Describe RuvB
Helicase that catalyzes branch migration
hexamer = binds as ring around DNA
37kDa
Describe RuvC
Nuclease which resolves Holliday structures
19kDa
Why may gene conversion come about?
Via general recombination and DNA repair mechanisms
Mismatch repair = replace mismatch DNA with copy of complementary strand
What is the mechanism of gene conversion?
Mismatch repair excises portion of green strand
DNA synthesis fills gap = creating an extra copy of the red allele of gene X
DNA replication = both chromosomes carry the red allele of gene X
What are the site-specific recombination ‘methods’?
Transposons
Phage integration and excision
Cre-LoxP system
What enzyme is required for DNA-only transposons?
Transposase
What enzyme is required for retroviral-like retrotransposon?
Reversetranscriptase
Integrase
What enzyme is required for non-retroviral retrotransposons?
Reverse transcriptase
Endonuclease
What is DNA-only transposon movement?
Cut and paste mechanism = excised from one spot on genome and inserted into another
Where are DNA-only transposons found and what are they responsible for?
Predominately in bacteria
Responsible for spread of antibiotic resistance in bacterial strains
Describe transposase function and structure
Dimer, each monomer recognizes the same specific DNA sequence at the ends of transposons = short inverted repeat sequences
What is the mechanism of cute-and-paste transposition?
Transposon in donor chromosome
Trasposase monomers recognize short inverted repeat sequences
Transposome formed = complex between enzyme and DNA
Target chromosome has staggered cuts made
DNAp and ligase integrate transposon from donor into recipient
Flanking direct repeats need to be generated
When is retroviral-like retrotransposons used?
Some viruses use transpositional site-specific recombination to move themselves into host chromosomes
What is retroviral-like retrotransposons mode of movement?
Moves via RNA intermediate
What is RNA intermediate produced by?
Promoter in long terminal repeats (LTR)
What is the mechanism of retroviral-like retrotransposons?
Viral entry into cell adn loss fo envelope
Reverse transcriptase makes RNA then makes DNA
Integration of DNA copied into host chromosome = with integrase
Transcription and translation = making new virus particles
What is the mode of movement in non-retroviral retrotransposons?
Move via RNA copy = often produced from neighbouring promoter
What is the mechanism of non-retroviral retrotransposons?
Donor DNA copied to RNA
Synthesis of reverse transcriptase/endonuclease
Binds to the RNA
Cleavage of target DNA by nuclease
DNA-primed reverse transcription
Multistep pathway produces second DNA strand
What is Gateway Cloning?
t
What is the BP reaction?
Gene + donor vector
Integrate gene into vector = needs BP clonase, integrase and integration host factor
Creates entry clone and by-product
What is the LR reaction?
Entry clone + destination vector
Excision of gene to be put into destination vector = needs Int, IHF and excisionase
Creates expression clone and toxic by-product
What is Cre and its function?
Bacteriophage P1 integrase
Catalyzes site-specific combination between loxP sites
What can site-specific recombination be used for and how?
Regulate gene expression
