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Genetics III > Lecture 6 - Site specific recombination > Flashcards

Lecture 6 - Site specific recombination Flashcards

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1
Q

What does a site specific recombination system comprise?

A

recombinase
recombination substrates
any accessory proteins or sites that are required to control the outcome

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2
Q

What do integrases, resolvases and invertases do?

A

Integrases - mediate integration into and excision of DNA out of host chromosome. e.g. integration occures when virus enter lysogenic phase and excision when phage exits lysogeny
Resolvases - resolve
Invertases - Invert DNA

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3
Q

What are the properties of conservative site specific recombination reactions?

A
  • no energy is lost during DNA breakage and rejoining
  • no need for ATP or another high energy substrate
  • recombinant products are reciprocal
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4
Q

How can site specific recombination reactions be conservative?

A

cleavage of the DNA backbone concomitantly transfers the high energy phosphate bond to a tyrosine or serine in the recombinase active site, preserving the bond

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5
Q

Where are conservative site-specific recombination systems found?

A

mostly in prokaryotes

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6
Q

What are the two separate classes of site specific recombinases in bacteria?

A
  • Tyrosine (Y) recombinases - phage lambda integrase, Cre recombinase, FLP recombinase, some topoisomerases
  • Serine (S) recombinases - phiC3I intergrase, resolvases and invertases
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7
Q

What is the general pathway that SSR reactions follow?

A
  1. Site specific recombinases bind the substrastes at a single specific phosphodiester bond in each recombination site
  2. recombinases ogigomerise forming a tetrameter, to bring the two sites together to form a nucloprotein complex by protein:protein interactions (synapsis)
  3. DNA cleavage
  4. Strand exchange to bring the recombinant ends together
  5. rejoining of the DNA backbone to form recombinants
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8
Q

What is the process of the strand exchange process?

A
  1. In the correct synapse the recombinases are activated to cleave DNA and concomitantly form a covelent linkage between cleaved DNA end and recombinase sub uni
  2. isomerisation of the complex causes DNA ends to reorientate so that the ends that will ligate to form the recombinant products are in close proximity
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9
Q

What is the process following the injection of phage P1 genome into E.coli when the phage is in lysogeny?

A
  1. genomic DNA injected as linear chromosome. arrangement of loxP sites are in direct repeat at each end of the phage P1 genomic DNA
  2. Cre recombinase is expressed when phage P1 infects E.coli
  3. Cre recombinase brings two loxP sites together forming a recombination between them
  4. results in formation of circular phage chromosome and small fragment derived from ends of genome
  5. replicates as a low copy number plasmid instead of integrating into the chromosome
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10
Q

What determines whether the IoxP sites are in direct repeat?

A

-each IoxP site has 13 bp of inverted repeat DNA bound by monomers of Cre = Recombinase Binding Element
-between RBEs 8bp sequence = Spacer, determines the polarity of the sites
Two IoxP sites in DR will have the same spacer reading the same direction on the same strand

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11
Q

What is the recombination pathway for tyrosine recombinases e.g. cre/loxp system?

A
  1. Two monomers of cre bind to each RBE in each recombination site
  2. Four cre monomers come together to to form a tetrameter bringing together the two loxP sites in close proximitry = synpase
  3. First stand exchange occurs after cleavage, first pair are active and mediate the cross over of one strand of the DNA.
  4. The active site tyrosine attacks the phosphodiester backbone, transferring the phosphate to tyrosine forming a 3’ phosphotryrosyl bond and free 5’OH
  5. free 5’OH end in spacer peels away and base pairs with the free spacer strand from the opposite recombination site
  6. 5’OH attacks the phosphotyrosyl bond tranferring the phosphate back to the DNA to ligate the DNA strand, forming a holliday junction intermediate
  7. Isomerisation of holliday structure actives second pair of Cre monomers and same reaction is repeated
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12
Q

Why might temperate phages need to use integration and excision?

A
  • can enter lysogeny, where integrates (attB and attP) into host genome
  • or can enter lytic growth cycle, excision (attL and attR) from host genome form circular phage chromosome
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13
Q

What are the substrates involved in phage integration and excision?

A

attP - phage DNA integration site (POP’)
attB - bacterial DNA integration site (BOB’)
attL - half bacterial integration site and phage (BOP’)
attR - half phage integration site and bacterial (POB’)

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14
Q

What is the structure or attB and attP?

A

attB - three parts, B arm, spacer (O) and B’ arm

attP - P arm, spacer (O) and P’ arm

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15
Q

What proteins are required for phage integration?

A

Integration host factor (IHF) contacts attP site and attL and attR sites
Int

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16
Q

What is required for phage excision?

A

Int
IHF (contacts attP site and attL and attR sites)
Xis
Fis
Xis and Fis bind to overlapping sites in attP and attL sites

17
Q

How can lambda integrase bind to core sites and arm regions of attP?

A

Two binding motifs

  • contact core/crossover region
  • contact arm sites
18
Q

What is the purpose of accessory binding proteins and accessory binding proteins in phage?

A

To direct the DNA through a specific pathway brings the integrase bound to the core sites to tetramerise and activate recombination

19
Q

What are two other ways phages can use to integrate into host chromsomes?

A
  • hijack host chromosome resolution system (XerCD/dif system) e.g. cholera toxin
  • use serine recombinases
20
Q

What is Gateway?

A

-simple way to move gene of interest between different types of plasmid vectors (eurkaryotes expression, prokaryote expression)and clone DNA

21
Q

What is the Gateway process to clone DNA?

A

1) Mix PCR product flanked by inverted attB recognition sites with the entry vector which contains toxin gene between attP flanking sites, add BPclonase,
2) leads to GOI inserted into empty clone flanked by attL sites. Vector can be selected against using gene toxic in bacterial cell
3) mis with destination vector, add LR clonase to move GOI into mammalian vector = expression plasmid

22
Q

Why does the gateway process work?

A

because of specificity of sites and the efficiency of reation
-because destination vector contains toxic gene prevents growth of destination vectors and promotes growth of cell containing GOI in destination vector

23
Q

Give an example of a phage encoded serine integrase

A

phiC3 integrase

24
Q

What are the features of phage-encoded serine integrases?

A
  • integration requires integrase
  • excision requires integrase and a recombination directionality factor RDF
  • simple attP, attB, attL and attR (all 40bp)
25
Q

How is the mechanism of serine recombinases different to tyrosine?

A
  1. cuts all the sites at the same time, dsbreaks in both recombination substrates
  2. isomerisation step strands are exchanged via subunit exchange where one pair of recombinases rotate by 180
26
Q

What keeps the serine recombinase structure together?

A

interface between subunits are very hydrophobic

27
Q

What are integrases controlled by?

A

the attachment sites and accessory proteins (Xis or RDFs)

28
Q

What are invertases and resolvases controlled by?

A

dectated through the topology of their recombination sites

29
Q

What is the biological relevance of the Hin inversion switch?

A

toggles gene expression between two types of flagellin, H1 and H2 , help avoid immune surviellence in salmoella antigenetically different

30
Q

How does the Hin inversion sequence work?

A

if the promoter is reading towards the H2 flagelling and gene for expression of a repressor of the promotor for H1 -> H2 expressed and H1 repressed
If the inversion element is orientated away from the H2 flaggella gene, H1 is expressed as it is no longer repressed I

31
Q

In hin inversion what occur for recombination to occur?

A

a synapse must form between hix sites in indirect repeat

32
Q

What detemines the inversion of the hix sites?

A

the structure of the hix site synapse and the orientation of the recombination sires

33
Q

How does the Hix element invert?

A

two indirectly repeated repeated hix sites can be brought together to form an inversion synapse
two parent hix sites are brought together looping out the invertible element
staggered breaks in the hix sites cause recombination to swap half of one site for half of the other inverting the element

34
Q

What happens when hix sites are in direct repeat?

A

cannot form profucts
same synapse structure and same cleavage pattern of spacer results in recombination sites that have base pair mismatches which cannot be ligated

35
Q

What happens when hix sites are in direct repeat and the synapse structure is changed?

A

DNA has to pass over itself to align two hix sites results in recombinant prooducts but the invertible element has been deleted

36
Q

How has Hin solved the problem of insuring against deletion?

A
  1. synapse topology is controlled by Fis which binds to recombination enhancer on the DNA and to Hin to trap the DNA in a specific topology, where Hin is active

Hin/Fis synapse topology still be assembled if hix sites in direct repeat but recombination will not occur, and deletion cannot occur as synapse topology stops DNA passing over itself

37
Q

How are circular chromosomes resoved?

A

Circular chromosomes may homologously recombine between daughter chromosomes before cell division resulting in tandemly repeated chromosome dimers
XerCD/dif SSR system reolves chromosome dimers
XerC and XerD are tyrosine recombinases that bind to RBEs of dif site, XerCD activated by FtsK to recombine two daughter chromsoome

Genetics III (12 decks)

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