SiteSpecific Recombination

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Site-Specific Recombination
Transposition of DNA
Chapter 11

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The subject of this chapter
Introduce

• genetic processes that
• rearrange DNA sequences
• and thus lead to a more Dyn-amic genome structure

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Two classes of genetic recombi-nation

• Conservative site-specific recombi-nation (CSSR)

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Transpositional recombination
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OUTLINE

• Conservative Site-Specific
• Recombination
• Biological Roles of Site-Specific
• Recombination
• Transposition
• Examples of Transposable
• Elements and Their Regulation
• V(D)J Recombination

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Conservative Site-Specific Recombination
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a . Site-specific Recombination Occurs at
Specific DNA Sequences in the Target DNA
Conservative Site-Specific Recombination

• Recombination sites (where DNA exchange
  occurs)the segment of DNA that will be moved
  carries specific short sequence elements .

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an example ? integration
Conservative Site-Specific Recombination
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The three types of CSSR recombination
Conservative Site-Specific Recombination
Depends on the organization of the
recombination sites on the DNA molecule or
molecule that participate in recombination.
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Structures involved in CSSR
Conservative Site-Specific Recombination
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b. Site-specific recombinases cleave and rejoin
DNA using a covalent protein-DNA intermediate
Conservative Site-Specific Recombination

• Serine recombinases
• Thyrosine recombinases

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Covalent-intermediate mechanism used by the
serine and tyrosine
Conservative Site-Specific Recombination
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c. Serine recombinases introduce double-stranded
breaks in DNA and then swap strands to promote
recombination
Conservative Site-Specific Recombination

• The serine recombinases cleave all for strands
  prior to strand exchange. These double-stranded
  DNA breaks in the parental DNA generate four
  double-stranded DNA segments.

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Conservative Site-Specific Recombination
Recombination By A serine recombinase
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d. Tyrosine recombinases break and rejoin one
pair of DNA strands at a time
Conservative Site-Specific Recombination

• 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 stands.

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Conservative Site-Specific Recombination
Recombination By A tyrosine Recombinase
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e. Structure of tyrosine recombinases bound to
DNA reveal the mechanism of DNA exchange
Conservative Site-Specific Recombination

• Cre is an phage P1-encoded protein, functioning
  to circularize the linear phage genome during
  infection
• Cre is a tyrosine recombinase
• The recombination sites of Cre is lox sites.
  Cre-lox is sufficient for recombination

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Conservative Site-Specific Recombination
Cre recombinase
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Conservative Site-Specific Recombination
Mechanism of site-specific recombination by The
Cre recombinase
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Conservative Site-Specific Recombination
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Biological Roles of Site-Specific Recombination
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Some functions of site-specific recombination
Biological Roles of Site-Specific Recombination

• Many phage insert their DNA into the host
  chromosome during infection using this
  recombination mechanism
• Alter gene expression.
• inversion of a DNA segment can allow two
  alternative genes to be expressed
• Help Maintain the structural integrity of
  circular DNA molecules during cycles of DNA
  replication, homologous, recombination, and cell
  division

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Some general themes of site-specific recombination
Biological Roles of Site-Specific Recombination

• All reactions depend critically on the assembly
  of the recombinase protein on the DNA, and the
  bringing together of the two recombination sites
• For some recombination this assembly requires
  only the recombinase and its recognition sequence
  ,others requires accessory proteins including
  Architectural Proteins

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a. l integrase promotes the integration and
Excision of a Viral Genome into the Host Cell
Chromosome
Biological Roles of Site-Specific Recombination

• To integrate, the l integrase protein(l lnt)
  catalyzes recombination between two specific
  sites, known as the att, or attachment, sites.
  The attP site is on the phage DNA, and the attB
  site is in the bacterial chromosome.
• P for phage. B for bacteria.

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Important to the regulation of l integtation
is the highly asymmetric organization of the
attP and attB sites
Biological Roles of Site-Specific Recombination
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b. Phage l excision requires a new DNA-bending
protein
Biological Roles of Site-Specific Recombination

• l excise an additional architectural protein,
  this one phage-encoded is essential for excisive
  recombination.
• This protein Called Xis, binds to specific DNA
  sequences and introduces bends in the DNA. its
  dual action as a stimulatory cofactor for
  excision and an inhibitor of integration ensures
  that the phage genome will be free, and remain
  free, from the host chromosome when Xis is
  present.

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c. The Hin recombinase inverts a segment of DNA
allowing expression of alternative genes
Biological Roles of Site-Specific Recombination

• The Salmonella Hin recombinase inverts a segment
  of the bacterial chromosome to allow expression
  of two alternative sets of genes.
• An example of a class of recombination reactions
  common in bacteria.
• known as programmed rearrangements Hin inversion
  is used to help the bacteria evade the host
  immune system.

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The genes controlled by Hin inversion encode two
alternative forms of flagellin, the protein
component of the flegellar fillament. Flagella
are on the surface of the bacteria and thus a
common target for the immune system
Biological Roles of Site-Specific Recombination

• Salmonella
• (showing
• flegella) invading
• cultured human
• cells.

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The chromosomal region inverted by Hin is about
1,000 bp and is flanked by specific recombination
sites called hixL and hixR.
Biological Roles of Site-Specific Recombination
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d. Hin recombination requires a DNA enhancer
Biological Roles of Site-Specific Recombination

• Hin recombination requires a sequence in addition
  to the hix sites. This short (60 bp) sequence is
  an enhancer that stimulates the rate of
  recombination 1,000-fold.
• An example of enhancer sequences that stimulate
  transcription .

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e. Recombinases convert multimeric circular DNA
molecules into monomers
Biological Roles of Site-Specific Recombination

• The chromosomes of most bacteria, plasmids and
  some viral genomes are circular.
• During the process of homologous recombination,
  these circular DNA sometimes form dimers and even
  multimeric forms, which can be can be converted
  back into monomer by site specific recombination.
• Site-specific recombinases also called resolvases
  catalyze such a process.

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Biological Roles of Site-Specific Recombination
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Essential that the enzyme catalyze resolution but
not the reverse reaction The Xer recombinase is
one of these enzymes , Xer recombinase is a
tyrosine recombinase and catalyzes the
monomerization of bacterial chromosomes and of
many bacterial plasmids. a heterotetramer
containing two subunits of XerC and two subunits
of XerD. XerC and XerD recognize different DNA
sequence.
Biological Roles of Site-Specific Recombination
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Biological Roles of Site-Specific Recombination
Pathways for Xer-mediated Recombination At Dif.
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f. There are other mechanisms to direct
recombination to specific segments of DNA
Biological Roles of Site-Specific Recombination

• Mating type switching in yeast.

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Transposition
Transposition
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Transposition
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.
• Movement occurs through recombination between
  the DNA sequences at the ends of the transposons
  and a sequence in the host DNA with little
  sequence selectivity.

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transposons
Transposition
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a. Some genetic elements move to new chromosomal
locations by transposition
Transposition
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Transposition of a mobile genetic element to a
new site in host DNA, which occurs with or
without duplication of the element.
Transposition
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b.There are three principle classes of
transposable elements
Transposition

• DNA transposons
• Viral-like retrotransposons including the
  retrovirus, which are also called LTR
  retrotransposons
• Poly-A retrotransposons, also called nonviral
  retrotransposons

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Transposition
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c. DNA transposons carry a transposase gene,
flanked by recombination sites
Transposition

• Recombination sites are at the two ends of the
  transposon and are inverted repeated sequences
  varying in length from 25 to a few hundred bp.
• The recombinase responsible for transposition are
  usually called transposases or integrases.
• Sometimes they carry a few additional genes.
  Example, many bacterial DNA transposons carry
  antibiotic resistance gene.

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d. Transposons exist as both autonomous and
nonautonomous elements
Transposition

• Autonomous transposons carry a pair of terminal
  inverted repeats and a transposase gene function
  independently
• Nonautonomous transposons carry only the
  terminal inverted repeats need the transposase
  encoded by autonomous transposons to enable
  transposition

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e.Viral-like retrotransposons and retroviruses
carry terminal repeat sequences and two genes
important for recombination
Transposition

• Inverted terminal repeat sequences for
  recombinase binding are embedded within long
  terminal repeats (LTRs), being organized on the
  two ends of the elements as direct repeats.
• reverse transcriptase (RT), using an RNA template
  to synthesize DNA.
• integrase (the transposase)

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Transposition
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f.Poly-A retrotransposons look like genes
Transposition

• Do not have the terminal inverted repeats.
• On end is called 5 UTR (untranslated region),
  the other end is 3 UTR followed by a stretch of
  A-T base pairs called the poly-A sequence.
  Flanked by short target site duplication.
• Carry two genes. ORF1 encodes an RNA-binding
  proteins. ORF2 encodes a protein with both
  reverse transcriptase (RT) and endonuclease
  activity

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g. DNA transposition by a cut-and-paste mechanism
Transposition
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h. DNA transposition by a replicative
mechanism/replicative transposition
Transposition
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Transposition
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i. Viral-like Retrotransposons Retroviruses
move using an RNA intermediate
Transposition
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j. DNA transposases and retroviral integrases are
members of a protein superfamily
Transposition
a
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Transposition
Similarities of catalytic domains of
transposases and integrases
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k. Poly-A Retrotransposition move by a reverse
splicing mechanism
Transposition
Transposition Of a poly-A Retransposon By target
Site-primed Teverse Transcription.
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Transposition