Chapter 11 Site-Specific Recombination

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Chapter 11 Site-Specific Recombination
Transposition of DNA
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Two classes of genetic recombination

• Conservative site-specific recombination (CSSR)
• Transpositional recombination

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OUTLINE

1. Conservative Site-Specific Recombination
2. Biological Roles of Site-Specific Recombination
3. Transposition
4. Examples of Transposable Elements and Their
   Regulation
5. V(D)J Recombination

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Conservative Site-Specific Recombination(CSSR)

• CSSR is recombination between two defined
  sequence elements

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1-1 CSSR occurs at specific DNA sequences in the
target DNA

• CSSR can generate three different types of DNA
  rearrangements
• 1.Insertion
• 2.Deletion
• 3.Inversion

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Structures involved in CSSR
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1-2 Site-specific recombinases cleave and rejoin
DNA using a covalent protein-DNA intermediate

• Therere two families of conservative
  site-specific recombinases
• 1. Serine Recombinases
• 2. Tyrosine Recombinases

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1-3 Serine recombinases introduce double-stranded
breaks in DNA and then swap strands to promote
recombination

• First , the serine recombinases cleave all four
  strands
• Second, DNA swap occurs
• Finally, the serine recombinases are liberated
  and they seal the DNA strands

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Recombination by a serine recombinase
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1-4 Tyrosine recombinases break and rejoin one
pair of DNA strands at a time

• 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.

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Recombination by a tyrosine recombinase
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1-5 Structure of tyrosine recombinases bound to
DNA reveal the mechanism of DNA exchange

• Cre is an enzyme encoded by phage P1 , which
  functions to circularize the linear phage genome
  during infection
• The recombination sites of Cre on the DNA are
  called lox sites.
• Only Cre protein and the lox sites are needed for
  complete recombination

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Biological roles of site-specific recombination

• 2-1 l integrase promotes the integration and
  Excision of a Viral Genome into the Host Cell
  Chromosome

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• Bacteriophage l infects a host bacterium and
  would establish a lysogen ,which requires the
  integration of phage DNA into host chromosome
• To integrate, lInt catalyzes recombination
  between two specific sitesattachment (att) sites
• attP site is on the phage DNA and attB site is
  on the bacterial genome
• lInt is a tyrosine recombinase, and the
  mechanism of strand exchange follows the pathway
  described above for the Cre protein

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The highly asymmetric organization of the
attPand attB sites is important to the regulation
of l integration

• The following figure showing
• recombination sites involved in l integration
  and excision showing the important sequence
  element

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2-2 Phage l excision requires a new DNA-binding
protein

• Phage l excision requires an architectural
  protein called Xis, which is phage-encoded
• Xis binds to the integrated attR sites to
  stimulate excision and to inhibit integration

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2-3 The Hin recombinase inverts a segment of DNA
allowing expression of alternative genes

• The Salmonella Hin recombinase inverts a segment
  of the bacterial chromosome to allow expression
  of two alternative sets of genes
• Hin recombinase is an example of programmed
  rearrangements in bacteria
• In the case of Hin inversion,recombination is
  used to help the bacteria evade the host immune
  system
• Hin is a serine recombinase which promotes
  inversion

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2-4 Hin recombination requires a DNA enhancer

• Hin recombination requires a DNA enhancer in
  addition to the hix sites
• Enhancer function requires the bacterial Fis
  protein
• the enhancer-Fis complex activates the catalytic
  steps of recombination
• Hin-catalyzed inversion is not highly regulated,
  rather, inversion occurs stochastically

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2-5 Recombinase converts multimeric circular DNA
molecules into monomers

• circular DNA molecules sometimes form dimers and
  even higher multimeric forms during the process
  of homologous recombination
• Site-specific recombinases (sometimes called
  resolvases) can resolve dimers and larger
  multimers into monomers

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Circular DNA molecules can form multimers
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• Xer recombinase is a tyrosine
• Xer catalyzes the monomerization of bacterial
  chromosomes and of many bacterial plasmids
• Xer is a heterotetramer, containing two subunits
  of XerC and two subunits of XerD
• XerC and XerD recognize different sequence
• The directional regulation of Xer-mediated
  recombination is achieved through the interaction
  between the Xer recombinase and a cell diversion
  protein called FtsK

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Pathways for Xer-mediated recombination at dif
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2-6 There are other mechanisms to direct
recombination to specific segaments of DNA

• The gene rearrangements responsible for assembly
  of gene segments encoding critical proteins for
  the vertebrate immune systemknown as V(D)J
  recombinationalso occurs at specific sites

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Transposition

• 3-1 Some genetic elements move to new
  chromosomal locations by transposition
• Transposition is a specific form of genetic
  recombination that moves certain genetic elements
  from one DNA site to another
• These mobile genetic elements are called
  transposable elements or transposons

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Transposition of a mobile genetic element to a
new site in host DNA
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• The transposons can insert within genes or
  regulatory sequence of a gene, which results in
  the completely disruption of gene function
• They can also insert within the regulatory
  sequences of a gene where their presence may lead
  to shanges in how that gene is expressed
• 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

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3-2 There are three principle classes of
transposable elements

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• 3-3 The recombinase responsible for
  transposition are usually called transposases or
  ,sometimes,integrases
• DNA transposons carry a transposase gene, flanked
  by recombination sites
• DNA transposons carry a gene encoding their own
  transposase, sometimes they may carry a few
  additional genes
• 3-4 Transposons exist as both autonomous and
  nonautonomous elements(Autonomous transposons and
  Nonautonomous transposons)

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• 3-5 Viral-like retrotransposons and
  retroviruses carry terminal repeat sequences and
  two genes important for recombination
• Viral-like retrotransposons and retroviruses
  carry LTRs
• Viral-like retrotransposons encode two proteins
  needed for their mobility integrase and reverse
  transcriptase (RT)
• 3-6 Poly-A retrotransposons look like genes

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3-7 DNA transposition by a cut-and-paste
mechanism

• The movement of a DNA transposon by a
  non-replicative mechanism called cut-and-paste
  transposition
• 1.First , transposase binds to the terminal
  inverted repeats at the end of the transopon and
  brings the two ends of the transopon DNA together
  to generate a stable protein-DNA complex called
  the synaptic complex or transpososome

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• 2.Next, the transopon DNA is excised from its
  original location in the genome
• 3.Then, the 3-OH ends of the transopon DNA
  attack the DNA phosphodiester bonds at the site
  of the new insertion, this DNA segment is called
  the target DNA
• 4.At last, the transopon DNA is covalently joined
  to the DNA at the target site by DNA strand
  tranfer. This reaction introduced a nick into the
  target DNA

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The cut-and-paste mechanism of transposition
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• The intermediate in cut-and-paste transposition
  is finished by gap repair
• 1.Two introduced nick are filled by a DNA repair
  polymerase(encoded by the host cell) and a DNA
  ligase
• 2.Filling in the gap gives rise to the target
  site duplications that flank transposons

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• There are multiple mechanisms for cleaving the
  nontransferred strand during DNA transposition
• 1.An enzyme other than tranposase can be used to
  cleave the nontransfered strand
• 2.The ranposase itself cleave the nontransfered
  strand by using an unusual DNA transesterification
  mechanism
• 3.DNA cleavage via a transesterification reaction
  can also occur between two ends of the transposon

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3-8 DNA transposition by a replicative mechanism

• First, the transposase protein is assembled on
  the two ends of the transposon DNA to generate a
  transpososome
• Then, DNA is cleaved at the ends of the
  transposon DNA

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• Then, the 3OH ends of the trsnsposon DNA are
  joined to the target sites by the DNA strand
  transfer reaction, which generate a doubly
  branched DNA molecule
• At last, The two DNA branches within this
  intermediate have the structure of a replication
  fork, and the DNA synthesis is proceeded
• This replication reaction generates two copies
  of the transposon DNA

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3-9 Viral-like retrotransposons and retroviruses
move using an RNA intermediate

• Recombination for retroelements involves an RNA
  intermediate
• A cycle of transposition starts with
  transcription of the retrotransposon (or
  retroviral) DNA sequence into RNA by cellular RNA
  polymerase. Transcription initiates at a promoter
  sequence within one of the LTRs.
• The RNA is then reverse transcribed to generate
  the cDNA

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• 3. The cDNA is recognized by Integrase and
  recombinate with a new target DNA site
• 4. Integrase assembles on the ends of this cDNA
  and cleaves a few nucleotides off the 3 ends of
  each strand
• 5. Integrase catalyzes the insertion of cleaved
  3 ends into a DNA target site in the host cell
  genome using the DNA strand transfer reaction.
• 6. Gap repair reaction generates target-site
  duplications.

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Mechanism of retroviral integration and
transposition of viral-like retrotransposons
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3-10 DNA transposases and retroviral integrases
are members of a protein superfamily

• Many different tranposases and integrases carry a
  catalytic domain that has a common
  three-dimensional shape
• This domain contains two D and a E
• The tranposase/integrase proteins use this same
  site to catalyze both the DNA cleavage and the
  DNA strand transfer
• Tranposases and integrases are only active when
  assembled into a synaptic complex, also called a
  transpososome, on DNA

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3-10 Poly-A Retrotransposition move by a reverse
splicing mechanism

• The Poly-A Retrotransposons use an RNA
  intermediate but use a mechanism different than
  that used by the viral-like elements. This
  mechanism is called target site primed reverse
  transcription
• 1. First, the DNA of an integrated element
  is transcripted by a cellular RNA polymerase
• 2. Then, newly synthesized RNA is exported
  to cytoplasm to produce ORF1 and ORF2 proteins

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• 3. The protein-RNA complex then reenters the
  nuclease and associates with the cellular DNA
• 4.The endonuclease initiates the intergration
  reaction by introducing a nick in the chromosomal
  DNA
• 5. The 3OH DNA end generated by the nicking
  action then serves as the primer for reverse
  transcription of the element RNA

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Transposition of a poly-A retrotransposon by
target site-primed reverse transcription
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Examples of transposable enements and their
regulation

• Two types of regulation appear as recurring
  themes
• Trnasposons control the number of their copies
  present in a given cell
• Trnasposons control target site choice

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4-1 IS4-family transposons are compact elements
with multiple mechanisms for copy number control

• Tn10 transposes via the cut-and paste mechanism,
  using the DNA hairpin strategy to cleave the
  nontransfered strands
• Tn10 limits its copy number in any given cell by
  strategies that restrict its transposition
  frequency. One mechanism is the use of an
  antisense RNA to control the expression of the
  transposase gene
• By this mechanism, cells that carry more copes of
  Tn10 will transcribe more of the antisense RNA,
  which in turn will limit expression of the
  transposase gene. The transposition
  frequencywill,therefore, be very low in such a
  strain

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Antisense regulation of Tn10 expression
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4-2 Transposition is coupled to cellular DNA
replication

• Bacteria methylate their DNA at GATC sites and
  GATC sites are hemimethylated for a few minutes
• It is during the brief periodwhen the Tn10 DNA
  is hemimethylatedthat transposition is more
  likely to occur
• Both RNA polymerase and transposase bind more
  tightly to the hemimethylated sequences than to
  their fully methylated versions. As a result,
  when the DNA is hemimethylated, the transposase
  gene is most efficiently expressed, and the
  transposaseprotein binds most efficiently to the
  DNA

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Transposition of Tn10 after passage of a
replication fork
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V(D)J recombination

• The principal mechanism cells use to generate
  antibodies and T cell receptors with such
  diversity relies on a specialized set of DNA
  rearrangement reactions known as V(D)J
  recombination

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Overview of the process of V(D)J recombination
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The early events in V(D)J recombination occur by
a mechanism similar to transposon excision

• Revombination sequences, called recombination
  signal sequences, flank the gene segments that
  are assembled by V(D)J recombination
• Recombination always occurs between a pair of
  recombination signal sequences which are
  organized as inverted repeats flanking the DNA
  segments that are destined to be joined

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• First, reconbinase recognizes the recombination
  signal sequences and pairs the two sites to form
  a protein-DNA synatic complex
• Then, the RAG1 proteins within this complex
  introduce single-stranded breaks in the DNA at
  each of the junctions between the recombination
  signal sequence and the gene segment that will be
  rearranged
• Then, this 3OH DNA end attacks the opposite
  strand of the DNA double helix,which results in
  the generation of a hairpin DNA end

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The V(D)J recombination pathway
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