Welcome Each of You to My Molecular Biology Class

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Welcome Each of You to My Molecular Biology Class
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Molecular Biology of the Gene, 5/E --- Watson et
al. (2004)
Part I Chemistry and Genetics Part II
Maintenance of the Genome Part III Expression
of the Genome Part IV Regulation Part V Methods
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Part II Maintenance of the Genome
Dedicated to the structure of DNA and the
processes that propagate, maintain and alter it
from one cell generation to the next
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Ch 6 The structures of DNA and RNA Ch 7
Chromosomes, chromatins and the nucleosome Ch 8
The replication of DNA Ch 9 The mutability and
repair of DNA Ch 10 Homologous recombination at
the molecular level Ch 11 Site-specific
recombination and transposition of DNA
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• Molecular Biology Course

• Chapter 11
• Site-Specific Recombination Transposition of DNA

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Although DNA replication, repair, homologous
recombination occur with high fidelity to ensure
the genome identity between generations, there
are genetic processes that rearrange DNA
sequences and thus lead to a more dynamic genome
structure
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• Two classes of genetic recombination for DNA
  rearrangement
• Conservative site-specific recombination (CSSR)
  recombination between two defined sequence
  elements
• Transpositional recombination (Transposition)
  recombination between specific sequences and
  nonspecific DNA sites

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Figure 11-1
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OUTLINE

• Conservative Site-Specific Recombination
• Biological Roles of Site-Specific Recombination
  (l phage integration/excision, multimeric genome
  resolution)
• Transposition ( concepts, learning from B.
  McClintock, DNA tranposons. Viral-like
  retrotransposons/retroviruses, poly-A
  retrotransposons)

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Topic 1 Conservative Site-Specific Recombination

• Exchange of non-homologous sequences at specific
  DNA sites(what)
• Mediated by proteins that recognize specific DNA
  sequences. (how)

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Conservative Site-Specific Recombination
1-1 Site-specific recombination occurs at
specific DNA sequences in the target DNA

• CSSR (conserved site-specific recombination) is
  responsible for many reactions in which a defined
  segment of DNA is rearranged.

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CSSR can generate three different types of DNA
rearrangements

• Figure 11-3

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If the two sites at which recombination will take
place are oriented oppositely to one another in
the same DNA molecule then the site-specific
reacombination results in inversion of the
segment of DNA between the two recombination sites
recombination at inverted repeats causes an
inversion
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If the two sites at which recombination will take
place are oriented in the same direction in the
same DNA molecule, then the segment of DNA
between the two recombinogenic sites is deleted
from the rest of the DNA molecule and appears as
a circular molecule. Insertion is the reverse
reaction of the deletion
recombination at direct repeats causes a deletion
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• Figure 11-4 Structures involved in CSSR

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Conservative Site-Specific Recombination
1-2 Site-specific recombinases cleave and rejoin
(join) DNA using a covalent protein-DNA
intermediate

• Serine Recombinases
• Tyrosine Recombinases

Figure 11-5
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The covalent protein-DNA intermediate conserves
the energy of the cleaved phosphodiester bond
within the protein-DNA linkage, which allows the
cleaved DNA strands to be rejoined/resealed by
reversal of the the cleavage process This
mechanistic feature contributes the
conservative to the CSSR name, because every
DNA bond that is broken during the reaction is
resealed by the recombinase without consuming any
external energy.
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Conservative Site-Specific Recombination
1-3 Serine recombinases introduce double-stranded
breaks in DNA and then swap strands to promote
recombination
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Conservative Site-Specific Recombination
Figure 11-6
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Conservative Site-Specific Recombination
1-4 Tyrosine recombinases break and rejoin one
pair of DNA strands at a time
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Figure 11-7
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Conservative Site-Specific Recombination
1-5 Structure of tyrosine recombinases bound to
DNA reveal the mechanism of DNA exchange

• Cre is a tyrosine recombinase
• Cre is an phage P1-encoded protein, functioning
  to circularize the linear phage genome during
  infection
• The recombination sites of Cre is lox sites.
  Cre-lox is sufficient for recombination
• Read Box11-1 for Cre application

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Figure 11-8
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Topic 2 Biological roles of site-specific
recombination
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• Many phage insert their DNA into the host
  chromosome during infection using this
  recombination mechanism. Example l phage
• Alter gene expression. Example Salmonella Hin
  recombinase (the details are not discussed in the
  class)
• Maintain the structural integrity of circular DNA
  molecules during cycles of DNA replication.
  Example resolvase that resolves dimer to monomer

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The general themes of site-specific recombination

• All reactions depend critically on the assembly
  of the recombinase protein on the DNA and bring
  together of the two recombination sites
• Some recombination requires only the recombinase
  and its recognition sequence for such an
  assembly some requires accessory proteins
  including Architectural Proteins that bind
  specific DNA sequences and bend the DNA.

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2-1 2 l integrase works with IHF and Xis to
integrate/excise the phage genome into/from the
bacterial chromosome
Biological roles of site-specific recombination

• The outcome of l bacteriophage infection of a
  host bacterium
• Establishment of the lysogenic state requires
  the integration of phage DNA into host chromosome
• lytic growth is the growth stage of
  multiplication of the independent phage DNA that
  requires the excision of the integrated phage DNA
  from the host genome.

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• Figure 11-2 l genome integration. Recombination
  always occurs at exactly the same sequence within
  two recombination sites, one on the phage DNA,
  and the other on the bacterial DNA.

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Phage genome
Crossover regions
Bacterial genome
Int (l-encoded integrase) Xis (l-encoded
excisionase)
IHF (integration host factor encoded by bacteria)
Figure 11-9
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• l-encoded integrase (Int)
• catalyzes recombination between two attachment
  (att) sites. attP site is on the phage DNA and
  attB site is on the bacterial genome
• Is a tyrosine recombinase, and the mechanism of
  strand exchange is similar to that catalyzed by
  Cre recombinase.
• Requires accessory proteins to assemble the
  integrase on the att sites. Both IHF and Xis are
  architectural proteins. IHF binds to DNA to bring
  together the Int recognition sites. Xis binds to
  the integrated att sites to stimulate excision
  and to inhibit integration (see 2-2).

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2-5 Recombinase converts 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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Figure 11-14 Circular DNA molecules can form
multimers
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Xer recombinase catalyzes the monomerization of
bacterial chromosomes and of many bacterial
plasmids. Xer recombinase is a member of the
tyrosine recombinase family Xer is a
heterotetramer containing two subunits of XerC
and two subunits of XerD. Both XerC and XerD are
tyrosine recombinases but recognize different DNA
sequence. The recombination sites in bacterial
chromosomes, called dif sites have recognition
sites for both XerC and XerD.
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Figure 11-15
The dimer only resolves when XerD is activated by
the presence of FtsK
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Topic 3 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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• FIGURE 11-17 Transposition of a mobile genetic
  element to a new site in host DNA, which occurs
  with or without duplication of the element.

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• Because transposition has little sequence
  selectivity in their choice of insertion sites,
  the transposons can insert within genes or
  regulatory sequence of a gene, which results in
  the completely disruption of gene function or the
  alteration of the expression of a gene. These
  disruption leads to the discovery of transposable
  elements by Barbara McClintock.

Box 11-3 Example of corn cob showing color
variegation due to transposition
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• It was actually the ability of transposable
  elements to break chromosomes that first came to
  McClintock attention (late 1940s). She found that
  some maize (??) strains experienced frequent
  chromosome-broken, and the hotspots for
  chromosome breaks varied among different strains
  and among different chromosomal locations in the
  descendents (??) of an individual plant, which
  leads to the concept that genetic elements could
  move/transpose

plant genomes are very rich in functional
transposons
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Discovery of Transposition Barbara McClintock
In the fall of 1921 I attended the only
course in genetics open to undergraduate students
at Cornell University. It was conducted by C. B.
Hutchison, then a professor in the Department of
Plant Breeding, College of Agriculture, who soon
left Cornell to become Chancellor of the
University of California at Davis, California.
Relatively few students took this course and most
of them were interested in pursuing agriculture
as a profession. Genetics as a discipline had not
yet received general acceptance. Only twenty-one
years had passed since the rediscovery of
Mendel's principles of heredity. Genetic
experiments, guided by these principles, expanded
rapidly in the years between 1900 and 1921.
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The results of these studies provided a solid
conceptual framework into which subsequent
results could be fitted. Nevertheless, there was
reluctance on the part of some professional
biologists to accept the revolutionary concepts
that were surfacing. This reluctance was soon
dispelled as the logic underlying genetic
investigations became increasingly evident. When
the undergraduate genetics course was completed
in January 1922, I received a telephone call from
Dr. Hutchison. He must have sensed my intense
interest in the content of his course because the
purpose of his call was to invite me to
participate in the only other genetics course
given at Cornell. It was scheduled for graduate
students. His invitation was accepted with
pleasure and great anticipations. Obviously, this
telephone call cast the die for my future. I
remained with genetics thereafter.
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At the time I was taking the undergraduate
genetics course, I was enrolled in a cytology
course given by Lester W. Sharp of the Department
of Botany. His interests focused on the structure
of chromosomes and their behaviors at mitosis and
meiosis. Chromosomes then became a source of
fascination as they were known to be the bearers
of "heritable factors". By the time of
graduation, I had no doubts about the direction I
wished to follow for an advanced degree. It would
involve chromosomes and their genetic content and
expressions, in short, cytogenetics. This field
had just begun to reveal its potentials. I have
pursued it ever since and with as much pleasure
over the years as I had experienced in my
undergraduate days.
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After completing requirements for the Ph.D.
degree in the spring of 1927, I remained at
Cornell to initiate studies aimed at associating
each of the ten chromosomes comprising the maize
complement with the genes each carries. With the
participation of others, particularly that of Dr.
Charles R. Burnham, this task was finally
accomplished. In the meantime, however, a
sequence of events occurred of great significance
to me. It began with the appearance in the fall
of 1927 of George W. Beadle (a Nobel Laureate) at
the Department of Plant Breeding to start studies
for his Ph.D. degree with Professor Rollins A.
Emerson. Emerson was an eminent geneticist whose
conduct of the affairs of graduate students was
notably successful, thus attracting many of the
brightest minds. In the following fall, Marcus M.
Rhoades arrived at the Department of Plant
Breeding to continue his graduate studies for a
Ph.D. degree, also with Professor Emerson.
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Rhoades had taken a Masters degree at the
California Institute of Technology and was well
versed in the newest findings of members of the
Morgan group working with Drosophila. Both Beadle
and Rhoades recognized the need and the
significance of exploring the relation between
chromosomes and genes as well as other aspects of
cytogenetics. The initial association of the
three of us, followed subsequently by inclusion
of any interested graduate student, formed a
close-knit group eager to discuss all phases of
genetics, including those being revealed or
suggested by our own efforts. The group was
self-sustaining in all ways. For each of us this
was an extraordinary period. Credit for its
success rests with Professor Emerson who quietly
ignored some of our seemingly strange behaviors.
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Over the years, members of this group have
retained the warm personal relationship that our
early association generated. The communal
experience profoundly affected each one of
us. The events recounted above were, by far, the
most influential in directing my scientific life.
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• Born 1902, Brooklyn, New York
• B.A. 1923, Cornell University
• Ph.D. 1927, Cornell University, Botany
• 1927-1931, Instructor in Botany, Cornell
  University
• 1931-1933, Fellow, National Research Council
• 1933-1934, Fellow, Guggenheim Foundation
• 1934-1936, Research Associate, Cornell University
  
• 1936-1941, Assistant Professor, University of
  Missouri
• 1942-1967, Staff member, Carnegie Institution of
  Washington's Department of Genetics, Cold Spring
  Harbor, NY
• 1967-1992, Distinguished Service Member, CIW
  Department of Genetics, Cold Spring Harbor
• 1944, Member, National Academy of Sciences
• 1945, President, Genetics Society of America
• 1967, Kimber Medal
• 1970, National Medal of Science
• 1981, Lasker Award
• 1983, Nobel Prize in Physiology or Medicine

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You, CLS students of Wuhan University, get to
learn to enjoy science/to be interested in
science, but not to enjoy good scores.
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The biological relevance of transposons

• Transposons are present in the genomes of all
  life-forms. (1) transposon-related sequences can
  make up huge fractions of the genome of an
  organism (50 of human and maize genome). (2) the
  transposon content in different genomes is highly
  variable (Fig 11-18).

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2. The genetic recombination mechanisms of
transposition are also used for other functions
than the movement of transposons, such as
integration of some virus into the host genome
and some DNA rearrangement to alter gene
expression V(D)J recombination.
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3-(1-6) There are three principle classes of
transposable elements

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

Transposition
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FIGURE 11-19 Genetic organization of the three
classes of transposable elements
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3-2 DNA transposons carry a transposase gene,
flanked by recombination sites

• 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.

Transposition
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3-3 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 the terminal
  inverted repeats but not the functional
  transposase need the transposase encoded by
  autonomous transposons to enable transposition

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3-4 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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3-5 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. Truncated elements lacking complete 5
  UTR??

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3-(7-9) DNA transposition by a cut-and-paste
mechanism (non-replicative mechanism)

• Multimers of transposase binds to the terminal
  inverted repeats of the transposons, and bring
  two ends together to form a stable protein-DNA
  complex called the synaptic complex/transpososome.
  
• This complex ensures the DNA cleavage and joining
  reaction, which is called strand transfer and is
  similar to the recombinase

Transposition
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• The transposase first cleaves one DNA strand at
  each end of the transposon, resulting in free
  3-OH groups
• Different transposons use different mechanism to
  cleave the second strands, resulting in 5 ends
  at the transposons. The mechanism including using
  a secondary enzyme (Tn7), using an unusual DNA
  transesterification mechanism to generate a DNA
  hairpin structure subsequently resolved by
  transposases (Tn10, Tn5) (3-9, Fig 11-21)

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• The 3OH ends of the transposon attack the DNA
  phosphodiester bonds at the sites of the new
  insertion/target DNA, resulting in transposon
  insertion. This DNA rejoining reactions occurs by
  a one-step transesterification reaction called
  DNA strand transfer.
• The intermediate with two nicks is finished by
  gap repair. The old insertion site having a
  double-stranded break are repaired by DSB repair
  (3-8)

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FIGURE 11-20 The cut-and-paste mechanism of
transposition
One-step transesterification
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3-10 DNA transposition by a replicative
mechanism/replicative transposition

• The mechanism is similar to the cut-and-paste
  transposition.
• The assembly of the transposase protein on the
  two ends of the transposon DNA to generate the
  transpososome.
• The transposase first cleaves one DNA strand at
  each end of the transposon, resulting in two 3OH
  ends. BUT NO cleavage occurs at the second strand.

Transposition
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• The 3OH ends of the transposon DNA are then
  joined to the target sites by the DNA strand
  transfer reaction, resulting in a doubly branched
  DNA molecule.
• The two branches within this intermediate have
  the structure of a replication fork, which
  recruits the replication proteins for strand
  synthesis. As a result, the donor DNA is
  duplicated in the host DNA.
• Replicative transposition frequently causes
  chromosomal inversions and deletions that can be
  highly detrimental (???) to the host cell.

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FIGURE 11-22 Replicative transposition
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3-11 Viral-like Retrotransposons Retroviruses
move using an RNA intermediate

• The mechanism is similar to the DNA transposons
  (Cut-and-Paste). The major difference is the
  involvement of an RNA intermediate.
• Transcription of the retrotransposon (or
  retroviral) DNA sequence into RNA by cellular RNA
  polymerase, which is initiated at a promoter
  sequence within one of the LTRs.

Transposition
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• The RNA is then reverse transcribed to cDNA
  (dsDNA) that is free from any flanking host DNA
  sequences, resulting in the excised form of
  transposon
• Integrase assembles on the ends of cDNA and
  cleaves a few nucleotides off the 3ends,
  generating 3OHs.
• Integrase inserts the transposon into target site
  using the DNA strand transfer reaction.
• Gap repair and ligation complete the
  recombination and generate target-site
  duplications.

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Figure 11-23 Mechanism of retroviral integration
and transposition of viral-like retrotransposons.
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3-12 DNA transposases and retroviral integrases
are members of a protein superfamily
Transposition
MuA
Tn5
RSV integrase
FIGURE 11-24 Similarity of catalytic domains of
transposases and integrases. (a) structure of the
conserved core domains of three transposases and
intergrase
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FIGURE 11-24 Similarity of catalytic domains of
transposases and integrases. (b) Scematic of the
domain organization of the above three proteins
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3-13 Poly-A Retrotransposition move by a reverse
splicing mechanism

• Using an RNA intermediate but a different
  mechanism from that of the viral-like
  retrotransposons
• The mechanism used is called target site primed
  reverse transcription.
• Transcription of the integrated DNA
• The newly synthesized RNA is exported to
  cytoplasm to produce ORF1 and ORF2 proteins,
  which remain to bind the RNA

Transposition
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• The protein-RNA complex reenters the nuclease and
  associate with the chromosomal DNA
• The endonuclease activity of ORF2 introduce a
  nick on the chromosomal DNA at the T-rich sites.
• The 3OH generated on the target DNA serves as
  the primer for reverse transcription of the
  element RNA (ORF2)

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Key points

• Conservative Site-Specific Recombination
  (concept, three types, mechanisms-serine and
  tyrosine recombinases)
• Biological Roles of Site-Specific Recombination
  (l phage integration/excision, multimeric genome
  resolution)
• Transposition ( concepts, learning from B.
  McClintock, DNA tranposons. Viral-like
  retrotransposons/retroviruses, poly-A
  retrotransposons)