Welcome Each of You
to My Molecular
Biology Class
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
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
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
Chapter 11
Site-Specific Recombination
& Transposition of DNA
?Molecular Biology Course
There are genetic processes
that rearrange DNA
sequences and thus lead to
a more dynamic genome
structure
Two classes of genetic recombination
? Conservative site-specific
recombination (CSSR),
recombination between two defined
sequence elements
? Transpositional recombination
(Transposition),recombination
between specific sequences and
nonspecific DNA sites
Figure 11-1
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
Topic 1,Conservative Site-
Specific Recombination
1.Exchange of non-homologous
sequences at specific DNA
sites(what)
2.Mediated by proteins that
recognize specific DNA sequences,
(how)
? CSSR (conserved site-specific
recombination) is responsible
for many reactions in which a
defined segment of DNA is
rearranged,
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1-1 Site-specific recombination
occurs at specific DNA sequences in
the target DNA
CSSR can generate three
different types of DNA
rearrangements
Figure 11-3
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
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
Figure 11-4
Structures
involved in
CSSR
?Serine Recombinases
?Tyrosine Recombinases
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1-2 Site-specific recombinases
cleave and rejoin DNA using a
covalent protein-DNA intermediate
Figure 11-5
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1-3 Serine recombinases introduce
double-stranded breaks in DNA and
then swap strands to promote
recombination
Conservative Site-Specific Recombination
Figure 11-6
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1-4 Tyrosine recombinases break
and rejoin one pair of DNA strands at
a time
Figure 11-7
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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
Figure 11-8
Topic 2 Biological roles of
site-specific recombination
? 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
? Maintain the structural integrity of
circular DNA molecules during
cycles of DNA replication,Example,
resolvase that resolves dimer to
monomer
1,All reactions depend critically on the
assembly of the recombinase protein on
the DNA and bring together of the two
recombination sites
2,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.
The general themes of site-specific
recombination
2-1 l integrase promotes the integration and
Excision of a Viral Genome into the Host
Cell Chromosome
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.
Bacterial genome
Phage genome
Crossover regions
Int (l-encoded integrase)
Xis (l-encoded
excisionase)
IHF (integration host
factor encoded by
bacteria)
Figure 11-9
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),
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,which
is known as an example of
programmed rearrangements
common in bacteria.
Hin inversion is used to help the
bacteria evade the host immune
system.
Hin is a serine recombinase
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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.
By using Hin inversion to switch between
these two alternative forms,some
bacterial individuals can aviod
recognition by host immune system
Figure 11-11 Salmonella
(showing flegella)
invading cultured human
cells.
Figure 11-11 Hin inversion
fljB encodes H2 flagellin
fljA encodes a transcriptional
repressor of H1 flagellin
2-5 Recombinase converts multimeric
circular DNA molecules into monomers
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
Xer recombinase is a tyrosine
recombinase and 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 DNA sequence.
Figure 11-15
The dimer only resolves when XerD is activated by
the presence of FtsK
Topic 3 Transposition (转座 )
? 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.
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.
? 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
? It was actually the ability of
transposable elements to break
chromosomes that first came to
McClintock attention (late 1940s),She
found that some maize (玉米 ) strains
experienced frequent chromosome-
broken,and the,hotspots” for
chromosome breaks varied among
different strains and among different
chromosomal locations in the
descendents (后代 ) of an individual
plant,which leads to the concept that
genetic elements could
move/transpose
plant genomes are very rich in
functional transposons
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,
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.
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.
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,
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.
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.
?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
The biological relevance of transposons
1,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)
2,The genetic recombination mechanisms
of transposition are also used for other
functions,such as integration of some
virus into the host genome and some
DNA rearrangement to alter gene
expression [V(D)J recombination].
3-(1-6) There are three principle classes
of transposable elements
1,DNA transposons
2,Viral-like retrotransposons
including the retrovirus,which
are also called LTR
retrotransposons
3,Poly-A retrotransposons,also
called nonviral
retrotransposons.
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FIGURE 11-19 Genetic organization
of the three classes of transposable
elements
3-2 DNA transposons carry a transposase
gene,flanked by recombination sites
1,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.
2,The recombinase responsible for
transposition are usually called
transposases or integrases.
3,Sometimes they carry a few
additional genes,Example,many
bacterial DNA transposons carry
antibiotic resistance gene.
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3-3 Transposons exist as both
autonomous and nonautonomous
elements
1,Autonomous transposons,carry a
pair of terminal inverted repeats
and a transposase gene; function
independently
2,Nonautonomous transposons,
carry only the terminal inverted
repeats; 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
1,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.
2,reverse transcriptase (RT),using an
RNA template to synthesize DNA.
3,integrase (the transposase)
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3-5 Poly-A retrotransposons look like
genes
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1,Do not have the terminal inverted
repeats,
2,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.
3,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
3-(7-9) DNA transposition by a cut-and-
paste mechanism (non-replicative
mechanism)
1,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
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2,The transposase first cleaves one DNA
strand at each end of the transposon,
resulting in free 3’-OH groups
3,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)
4,The 3’OH 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.
5,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)
FIGURE 11-
20 The cut-
and-paste
mechanism
of
transposition
One-step
transesterification
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,
2,The transposase first cleaves one
DNA strand at each end of the
transposon,resulting in two 3’OH
ends,BUT NO cleavage occurs at the
second strand.
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3,The 3’OH 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.
4,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 (有害的 )
to the host cell.
FIGURE 11-22
Replicative
transposition
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.
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2,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
3,Integrase assembles on the ends of
cDNA and cleaves a few nucleotides
off the 3’ends,generating 3’OHs.
4,Integrase inserts the transposon into
target site using the DNA strand
transfer reaction.
5,Gap repair and ligation complete the
recombination and generate target-
site duplications.
Figure 11-23
Mechanism of
retroviral
integration
and
transposition
of viral-like
retrotranspos
ons.
3-12 DNA transposases and retroviral
integrases are members of a protein
superfamily
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FIGURE 11-24 Similarity of catalytic domains
of transposases and integrases,(a) structure
of the conserved core domains of three
transposases and intergrase
MuA Tn5 RSV integrase
FIGURE 11-24 Similarity of catalytic domains
of transposases and integrases,(b) Scematic
of the domain organization of the above three
proteins
3-13 Poly-A Retrotransposition move by
a,reverse splicing” mechanism
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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.
1,Transcription of the integrated DNA
2,The newly synthesized RNA is
exported to cytoplasm to produce
ORF1 and ORF2 proteins,which
remain to bind the RNA
3,The protein-RNA complex
reenters the nuclease and
associate with the chromosomal
DNA
4,The endonuclease activity of
ORF2 introduce a nick on the
chromosomal DNA at the T-rich
sites.
5,The 3’OH generated on the
target DNA serves as the primer
for reverse transcription of the
element RNA (ORF2)
That’s all for this chapter
to My Molecular
Biology Class
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
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
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
Chapter 11
Site-Specific Recombination
& Transposition of DNA
?Molecular Biology Course
There are genetic processes
that rearrange DNA
sequences and thus lead to
a more dynamic genome
structure
Two classes of genetic recombination
? Conservative site-specific
recombination (CSSR),
recombination between two defined
sequence elements
? Transpositional recombination
(Transposition),recombination
between specific sequences and
nonspecific DNA sites
Figure 11-1
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
Topic 1,Conservative Site-
Specific Recombination
1.Exchange of non-homologous
sequences at specific DNA
sites(what)
2.Mediated by proteins that
recognize specific DNA sequences,
(how)
? CSSR (conserved site-specific
recombination) is responsible
for many reactions in which a
defined segment of DNA is
rearranged,
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1-1 Site-specific recombination
occurs at specific DNA sequences in
the target DNA
CSSR can generate three
different types of DNA
rearrangements
Figure 11-3
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
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
Figure 11-4
Structures
involved in
CSSR
?Serine Recombinases
?Tyrosine Recombinases
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1-2 Site-specific recombinases
cleave and rejoin DNA using a
covalent protein-DNA intermediate
Figure 11-5
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1-3 Serine recombinases introduce
double-stranded breaks in DNA and
then swap strands to promote
recombination
Conservative Site-Specific Recombination
Figure 11-6
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1-4 Tyrosine recombinases break
and rejoin one pair of DNA strands at
a time
Figure 11-7
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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
Figure 11-8
Topic 2 Biological roles of
site-specific recombination
? 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
? Maintain the structural integrity of
circular DNA molecules during
cycles of DNA replication,Example,
resolvase that resolves dimer to
monomer
1,All reactions depend critically on the
assembly of the recombinase protein on
the DNA and bring together of the two
recombination sites
2,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.
The general themes of site-specific
recombination
2-1 l integrase promotes the integration and
Excision of a Viral Genome into the Host
Cell Chromosome
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.
Bacterial genome
Phage genome
Crossover regions
Int (l-encoded integrase)
Xis (l-encoded
excisionase)
IHF (integration host
factor encoded by
bacteria)
Figure 11-9
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),
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,which
is known as an example of
programmed rearrangements
common in bacteria.
Hin inversion is used to help the
bacteria evade the host immune
system.
Hin is a serine recombinase
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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.
By using Hin inversion to switch between
these two alternative forms,some
bacterial individuals can aviod
recognition by host immune system
Figure 11-11 Salmonella
(showing flegella)
invading cultured human
cells.
Figure 11-11 Hin inversion
fljB encodes H2 flagellin
fljA encodes a transcriptional
repressor of H1 flagellin
2-5 Recombinase converts multimeric
circular DNA molecules into monomers
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
Xer recombinase is a tyrosine
recombinase and 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 DNA sequence.
Figure 11-15
The dimer only resolves when XerD is activated by
the presence of FtsK
Topic 3 Transposition (转座 )
? 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.
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.
? 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
? It was actually the ability of
transposable elements to break
chromosomes that first came to
McClintock attention (late 1940s),She
found that some maize (玉米 ) strains
experienced frequent chromosome-
broken,and the,hotspots” for
chromosome breaks varied among
different strains and among different
chromosomal locations in the
descendents (后代 ) of an individual
plant,which leads to the concept that
genetic elements could
move/transpose
plant genomes are very rich in
functional transposons
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,
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.
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.
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,
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.
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.
?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
The biological relevance of transposons
1,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)
2,The genetic recombination mechanisms
of transposition are also used for other
functions,such as integration of some
virus into the host genome and some
DNA rearrangement to alter gene
expression [V(D)J recombination].
3-(1-6) There are three principle classes
of transposable elements
1,DNA transposons
2,Viral-like retrotransposons
including the retrovirus,which
are also called LTR
retrotransposons
3,Poly-A retrotransposons,also
called nonviral
retrotransposons.
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FIGURE 11-19 Genetic organization
of the three classes of transposable
elements
3-2 DNA transposons carry a transposase
gene,flanked by recombination sites
1,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.
2,The recombinase responsible for
transposition are usually called
transposases or integrases.
3,Sometimes they carry a few
additional genes,Example,many
bacterial DNA transposons carry
antibiotic resistance gene.
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3-3 Transposons exist as both
autonomous and nonautonomous
elements
1,Autonomous transposons,carry a
pair of terminal inverted repeats
and a transposase gene; function
independently
2,Nonautonomous transposons,
carry only the terminal inverted
repeats; 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
1,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.
2,reverse transcriptase (RT),using an
RNA template to synthesize DNA.
3,integrase (the transposase)
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3-5 Poly-A retrotransposons look like
genes
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1,Do not have the terminal inverted
repeats,
2,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.
3,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
3-(7-9) DNA transposition by a cut-and-
paste mechanism (non-replicative
mechanism)
1,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
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2,The transposase first cleaves one DNA
strand at each end of the transposon,
resulting in free 3’-OH groups
3,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)
4,The 3’OH 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.
5,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)
FIGURE 11-
20 The cut-
and-paste
mechanism
of
transposition
One-step
transesterification
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,
2,The transposase first cleaves one
DNA strand at each end of the
transposon,resulting in two 3’OH
ends,BUT NO cleavage occurs at the
second strand.
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3,The 3’OH 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.
4,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 (有害的 )
to the host cell.
FIGURE 11-22
Replicative
transposition
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.
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2,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
3,Integrase assembles on the ends of
cDNA and cleaves a few nucleotides
off the 3’ends,generating 3’OHs.
4,Integrase inserts the transposon into
target site using the DNA strand
transfer reaction.
5,Gap repair and ligation complete the
recombination and generate target-
site duplications.
Figure 11-23
Mechanism of
retroviral
integration
and
transposition
of viral-like
retrotranspos
ons.
3-12 DNA transposases and retroviral
integrases are members of a protein
superfamily
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FIGURE 11-24 Similarity of catalytic domains
of transposases and integrases,(a) structure
of the conserved core domains of three
transposases and intergrase
MuA Tn5 RSV integrase
FIGURE 11-24 Similarity of catalytic domains
of transposases and integrases,(b) Scematic
of the domain organization of the above three
proteins
3-13 Poly-A Retrotransposition move by
a,reverse splicing” mechanism
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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.
1,Transcription of the integrated DNA
2,The newly synthesized RNA is
exported to cytoplasm to produce
ORF1 and ORF2 proteins,which
remain to bind the RNA
3,The protein-RNA complex
reenters the nuclease and
associate with the chromosomal
DNA
4,The endonuclease activity of
ORF2 introduce a nick on the
chromosomal DNA at the T-rich
sites.
5,The 3’OH generated on the
target DNA serves as the primer
for reverse transcription of the
element RNA (ORF2)
That’s all for this chapter
