The second section   of recombination systems S ite-Specific Recombination  &  Transposition  By: Hatice Nur Halipçi
OUTLINE <ul><li>Site-Specific Recombination </li></ul><ul><li>Biological Roles of Site-Specific Recombination </li></ul><u...
Two classes of genetic recombination <ul><li>S ite-specific recombination (SSR) </li></ul><ul><li>Transpositional recombin...
Site-Specific Recombination   (SSR) <ul><li>SSR  is  recombination between two defined sequence elements </li></ul>
1-1 SSR occurs at  specific DNA sequences  in the target DNA <ul><li>SSR can generate three different types of DNA rearran...
I nsertion  is the addition of one   or more  nucleotide   base pairs  into a  DNA  sequence  Deletion  (also called  defi...
Structures involved in SSR
1-2  Site-specific recombinases cleave and rejoin DNA  <ul><li>There’re two families of conservative site-specific recombi...
1-3 Serine recombinases introduce double-stranded breaks in DNA and then swap strands to promote recombination <ul><li>Fir...
Recombination by a serine recombinase
1-4 Tyrosine recombinases break and rejoin one pair of DNA strands at a time <ul><li>In contrast to the serine recombinase...
Recombination by a tyrosine recombinase
Biological roles of site-specific recombination <ul><li>2-1    integrase  promotes the  integration and Excision  of a Vi...
<ul><li>Bacteriophage    infects a host bacterium and would establish a  lysogen  ( staying in the host chromosome presen...
The highly asymmetric organization of the  attP   and attB sites is important to the regulation of    integration
The figure showing: recombination sites involved in  integration and excision showing the important sequence element
2-2  Phage   excision requires a new DNA-binding protein <ul><li>Phage   excision requires an architectural protein ca...
2-3   The Hin recombinase   inverts a segment of DNA allowing expression of alternative genes <ul><li>The  Salmonella Hin ...
2-4 Hin recombination requires a DNA enhancer <ul><li>Hin recombination requires a DNA enhancer  in addition to the hix si...
2-5  Recombinase   converts multimeric circular DNA molecules into monomers <ul><li>C ircular DNA molecules sometimes form...
Circular DNA molecules can form multimers
<ul><li>Xer recombinase is a tyrosine  (site-spesific recombinase) </li></ul><ul><li>Xer catalyzes the monomerization of b...
Pathways for Xer-mediated recombination
Transposition <ul><li>3-1  Some  genetic elements move to new chromosomal locations by transposition </li></ul><ul><li>Tra...
Transposition of a mobile genetic element to a new site in DNA
<ul><li>The transposons can insert within genes or regulatory sequence of a gene, which results in the completely disrupti...
<ul><li>The recombinase responsible for transposition are usually called   transposases   or ,sometimes, integrases </li><...
Types of Transposition <ul><li>Cut and paste Transposition ( Conservative ) </li></ul><ul><li>Replicative transposition </...
C ut-and- P aste  transposition  (conservative) <ul><li>In  cut-and-paste  transposition , an element is cut out of one </...
Replicative transposition <ul><li>In  replicative transposition , an element is replicated, and one </li></ul><ul><li>copy...
Retrotransposition In  retrotransposition ,  t he element makes an RNA copy of itself which is  reversed-transcribed  into...
3- 2   There are three principle classes of transposable elements
<ul><li>·   DNA transposons  carry  terminal inverted repeats at both sites ( recognize recombinase) </li></ul><ul><li>·  ...
<ul><li>Viral-like retrotransposons  and retroviruses carry  terminal repeat sequences  and two genes important for recomb...
<ul><li>Poly-A retrotransposons  look like genes </li></ul><ul><li>They carry  5’ UTR ve 3’ UTR  (no translation here) </l...
<ul><li>B acteriophage - Mu </li></ul><ul><li>a model transposable element   </li></ul>
Temperate bacteriophage Mu (Mu - mutator) <ul><li>Upon infection of its E.coli host, phage Mu integrates by conservative t...
The biochemistry of Mu transposition <ul><li>The transposase is a protein called  MuA.  It has both Dna binding and endonu...
THANK YOU! THE END
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H.Nur Halipçi-genetics of recombination

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H.Nur Halipçi-genetics of recombination

  1. 1. The second section of recombination systems S ite-Specific Recombination & Transposition By: Hatice Nur Halipçi
  2. 2. OUTLINE <ul><li>Site-Specific Recombination </li></ul><ul><li>Biological Roles of Site-Specific Recombination </li></ul><ul><li>Transposition </li></ul><ul><li>Phage Mu- a model transposable element </li></ul>
  3. 3. Two classes of genetic recombination <ul><li>S ite-specific recombination (SSR) </li></ul><ul><li>Transpositional recombination </li></ul>
  4. 4. Site-Specific Recombination (SSR) <ul><li>SSR is recombination between two defined sequence elements </li></ul>
  5. 5. 1-1 SSR occurs at specific DNA sequences in the target DNA <ul><li>SSR can generate three different types of DNA rearrangements: </li></ul><ul><li>1.Insertion </li></ul><ul><li>2.Deletion </li></ul><ul><li>3.Inversion </li></ul>
  6. 6. I nsertion is the addition of one or more nucleotide base pairs into a DNA sequence Deletion (also called deficiency ) is a mutation in which a part of a chromosome or a sequence of DNA is missing. Inversion is a chromosome rearrangement in which a segment of a chromosome is reversed end to end, shortly reversing the orientation of a chromosomal segment.
  7. 7. Structures involved in SSR
  8. 8. 1-2 Site-specific recombinases cleave and rejoin DNA <ul><li>There’re two families of conservative site-specific recombinases: </li></ul><ul><li>1. Serine Recombinases </li></ul><ul><li>2. Tyrosine Recombinases </li></ul>
  9. 9. 1-3 Serine recombinases introduce double-stranded breaks in DNA and then swap strands to promote recombination <ul><li>First , the serine recombinases cleave all four strands </li></ul><ul><li>Second, DNA swap occurs </li></ul><ul><li>Finally, the serine recombinases are liberated and they seal the DNA strands </li></ul>
  10. 10. Recombination by a serine recombinase
  11. 11. 1-4 Tyrosine recombinases break and rejoin one pair of DNA strands at a time <ul><li>In contrast to the serine recombinases ,the tyrosine recombinases cleave and rejoin two DNA strands first, and only then cleave and rejoin the other two strands. </li></ul>
  12. 12. Recombination by a tyrosine recombinase
  13. 13. Biological roles of site-specific recombination <ul><li>2-1  integrase promotes the integration and Excision of a Viral Genome into the Host Cell Chromosome </li></ul>
  14. 14. <ul><li>Bacteriophage  infects a host bacterium and would establish a lysogen ( staying in the host chromosome presently without harming ) ,which requires the integration of phage DNA into host chromosome </li></ul><ul><li>To integrate,  Int catalyzes recombination between two specific sites—attachment (att) sites </li></ul><ul><li>attP site is on the phage DNA and attB site is on the bacterial genome </li></ul><ul><li> Int is a type of tyrosine recombinase </li></ul>
  15. 15. The highly asymmetric organization of the attP and attB sites is important to the regulation of  integration
  16. 16. The figure showing: recombination sites involved in integration and excision showing the important sequence element
  17. 17. 2-2 Phage  excision requires a new DNA-binding protein <ul><li>Phage  excision requires an architectural protein called Xis , which is phage-encoded </li></ul><ul><li>Xis binds to the integrated attR sites to stimulate excision and to inhibit integration </li></ul>
  18. 18. 2-3 The Hin recombinase inverts a segment of DNA allowing expression of alternative genes <ul><li>The Salmonella Hin recombinase inverts a segment of the bacterial chromosome to allow expression of two alternative sets of genes </li></ul><ul><li>Hin recombinase is an example of programmed rearrangements in bacteria </li></ul><ul><li>In the case of Hin inversion,recombination is used to help the bacteria evade the host immune system </li></ul><ul><li>Hin is a serine recombinase which promotes inversion </li></ul>
  19. 19. 2-4 Hin recombination requires a DNA enhancer <ul><li>Hin recombination requires a DNA enhancer in addition to the hix sites </li></ul><ul><li>Enhancer function requires the bacterial Fis protein </li></ul><ul><li>the enhancer-Fis complex activates the catalytic steps of recombination </li></ul><ul><li>Hin-catalyzed inversion is not highly regulated, rather, inversion occurs stochastically </li></ul>
  20. 20. 2-5 Recombinase converts multimeric circular DNA molecules into monomers <ul><li>C ircular DNA molecules sometimes form dimers and even higher multimeric forms during the process of homologous recombination </li></ul><ul><li>Site-specific recombinases (sometimes called resolvases) can resolve dimers and larger multimers into monomers </li></ul>
  21. 21. Circular DNA molecules can form multimers
  22. 22. <ul><li>Xer recombinase is a tyrosine (site-spesific recombinase) </li></ul><ul><li>Xer catalyzes the monomerization of bacterial chromosomes and of many bacterial plasmids </li></ul><ul><li>Xer is a heterotetramer, containing two subunits of XerC and two subunits of XerD </li></ul><ul><li>XerC and XerD recognize different sequence </li></ul><ul><li>The directional regulation of Xer-mediated recombination is achieved through the interaction between the Xer recombinase and a cell diversion protein called FtsK </li></ul>
  23. 23. Pathways for Xer-mediated recombination
  24. 24. Transposition <ul><li>3-1 Some genetic elements move to new chromosomal locations by transposition </li></ul><ul><li>Transposition is a specific form of genetic recombination that moves certain genetic elements from one DNA site to another </li></ul><ul><li>These mobile genetic elements are called transposable elements or transposons </li></ul>
  25. 25. Transposition of a mobile genetic element to a new site in DNA
  26. 26. <ul><li>The transposons can insert within genes or regulatory sequence of a gene, which results in the completely disruption of gene function </li></ul><ul><li>They can also insert within the regulatory sequences of a gene where their presence may lead to c hanges in how that gene is expressed </li></ul><ul><li>Transposable elements are present in the genomes of all life-forms. (1) transposon-related sequences can make up huge fractions of the genome of an organism. (2) the transposon content in different genomes is highly variable </li></ul>
  27. 27. <ul><li>The recombinase responsible for transposition are usually called transposases or ,sometimes, integrases </li></ul><ul><li>Transposons exist as both autonomous and nonautonomous elements </li></ul><ul><li>For example: IS10R is an autonomous element while IS10L is non-autonomous </li></ul><ul><li>N onautonomous transposons need a helper transposon to mobilize </li></ul>
  28. 28. Types of Transposition <ul><li>Cut and paste Transposition ( Conservative ) </li></ul><ul><li>Replicative transposition </li></ul><ul><li>Retrotransposition </li></ul>
  29. 29. C ut-and- P aste transposition (conservative) <ul><li>In cut-and-paste transposition , an element is cut out of one </li></ul><ul><li>site in a chromosome and pasted into a new site. </li></ul>
  30. 30. Replicative transposition <ul><li>In replicative transposition , an element is replicated, and one </li></ul><ul><li>copy is inserted at a new site; one copy also remains at the original </li></ul><ul><li>site. </li></ul>
  31. 31. Retrotransposition In retrotransposition , t he element makes an RNA copy of itself which is reversed-transcribed into a DNA copy which is then inserted (cDNA )
  32. 32. 3- 2 There are three principle classes of transposable elements
  33. 33. <ul><li>· DNA transposons carry terminal inverted repeats at both sites ( recognize recombinase) </li></ul><ul><li>· These inverted repeats act as the substrates fo r recombination reactions mediated by the transposase </li></ul><ul><li>· They carry a gene encoding their own transposase, sometimes they may carry a few additional genes which useful for the host (resistance etc.) </li></ul>
  34. 34. <ul><li>Viral-like retrotransposons and retroviruses carry terminal repeat sequences and two genes important for recombination </li></ul><ul><li>Viral-like retrotransposons and retroviruses carry LTRs (long terminal repeats) </li></ul><ul><li>, </li></ul><ul><li>Viral-like retrotransposons encode two proteins needed for their mobility: integrase and reverse transcriptase (RT ) </li></ul>
  35. 35. <ul><li>Poly-A retrotransposons look like genes </li></ul><ul><li>They carry 5’ UTR ve 3’ UTR (no translation here) </li></ul><ul><li>And also carry an A-T sequence </li></ul><ul><li>ORF1 encodes the protein which binds RNA </li></ul><ul><li>ORF2 encodes the protein which activates reverse transcriptase and endonuclease </li></ul>
  36. 36. <ul><li>B acteriophage - Mu </li></ul><ul><li>a model transposable element </li></ul>
  37. 37. Temperate bacteriophage Mu (Mu - mutator) <ul><li>Upon infection of its E.coli host, phage Mu integrates by conservative transpositions </li></ul><ul><li>Mu genome is ~ 37 kb </li></ul><ul><li>In the Mu system, transposition is obligatory for replication an thus the phage genome is replicated only as a cointegrate structure </li></ul>
  38. 38. The biochemistry of Mu transposition <ul><li>The transposase is a protein called MuA. It has both Dna binding and endonuclease activities </li></ul><ul><li>MuA normally acts in a multiprotein complex with several accessory proteins </li></ul><ul><li>MuB is an activator of MuA and provides some degree of target-site selectivity </li></ul><ul><li>HU is an E. Coli non-sequence –specific DNA binding protein </li></ul>
  39. 39. THANK YOU! THE END
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