Lecture 8

Report
DNA Recombination
Homologous recombination (HR)
1. Precision: HR mediates exchange between DNA segments that share extensive
sequence homology. Exchange may can occur at any point between the
homologous region, although particular DNA sequences may influence
frequency of exchange.
2. Efficiency: whenever sufficiently long homologous sequences are brought
together in a single cell under appropriate conditions, the production of
recombinant sequence is more of a rule than exception.
3. Complexity: Several proteins are required in base paring and strand exchange.
It is a conserved cellular process.
Site-specific recombination (SSR)
1. Extensive homology not required. Limited to specific sites.
2. Requires system-specific proteins and is independent of HR.
Site-Specific Recombination (SSR)
1.
Conservative SSR (CSSR):
i) Recombination occurs at specific sites, within short sequence
identity.
ii) strand exchange occurs by precise breakage and joining events.
iii) no synthesis or loss of DNA sequences occur.
iv) recombination is reciprocal.
2.
Non-conservative or Transpositional:
i) no homology between the recombination sites
ii) DNA synthesis accompanies breakage and joining events.
iii) recombination is non-reciprocal.
Biological consequences of recombination
Integration/Excision system: lambda (λ) system
Lysogeny
attP + attB
IHF
Integrase (Int)
attL + attR
Prophage induction
IHF
attL + attR Integrase (Int) attP + attB
Xis (phage encoded)
Other phages such as phi80, P2, P4 and P22 also employ
CSSR integration/excision system
Biological consequences of recombination
Plasmid segregation: Stable plasmid maintenance requires that plasmid
copies efficiently distribute into daughter cells at cell division. High copy
plasmids are randomly distributed. Low copy plasmids have to use
specialized partitioning system. Both mechanisms require that the
presence of multimeric plasmids, which can be generated by homologous
recombination. These multimers must be minimized to monomers prior to
partitioning. P1 phage uses site-specific recombination system, Cre-lox, to
resolve multimers into monomers. Single protein bearing recombinase
activity, Cre, is required for the reversible recombination reaction.
lox
Cre
lox
Cre-lox
P1 phage has circular genome but linear genetic map.
Recombinase: Cre
DNA recombination substrate: loxP
34 bp (13 bp inverted repeats flank 8 bp asymmetric sequence)
ATAACTTCGTATA GCATACAT TATACGAAGTTAT
TATTGAAGCATAT CGTATGTA ATATGCTTCAATA
LoxP sequence
13-8-13
Biological consequences of recombination
Inversion system (The FLP-FRT system):
The 2-micron circular plasmid often found in yeast encodes a
CSSR system that can invert a large segment of plasmid. The
inversion changes the orientation of replicative forks within
the plasmid and thereby promotes amplification to high copy
number.
FRT
FLP
Rep fork
Rep fork
Rep fork
Rep fork
FLP-FRT
Recombinase: FLP
Recombination site: FRT
13-13-8-13
13-8-13
FRT: original
FRT
R-RS
Recombinase: R
Recombination site: RS
12-7-12
The recombination sites pair in same direction
Inversion
a b c d
a b c d
Deletion/ Integration
Exchange/ Translocation
Mutant sites
LE/ RE mutation
LE mutant
RE mutant
Spacer Mutation
lox511
A
ATAACTTCGTATA GCATACAT TATACGAAGTTAT
TATTGAAGCATAT CGTATGTA ATATGCTTCAATA
Reactions between mutant sites
LE mutant X RE mutant
LE+RE mutant + loxP
loxP X lox511
lox511 X lox511
lox511 X lox512
Transposons
Features.
1. Autonomous, may have a non-autonomous form.
2. Excise non-replicatively and re-insert elsewhere.
Maize transposons:
1. Activator/Dissociation (Ac/Ds)
2. Enhancer/Suppressor-mutator (En/Spm)
3. Mutator (Mu)
Maize Activator Element (Ac)
TIR
TIR
TTT CATCCC TA
AAAGTAGGGAT
CAGGGATGAAA
GT CCC TACTTT
Exon 1
Exon 2
Exon 3
E4
E5
4565 bp
mRNA
ORF (2421 b)
Outermost nucleotides of terminal inverted repeats are not complementary
Ds element
1. Simple Ds elements are deletion derivatives of Ac, which
have lost internal sequence for trans-acting factor.
2. Composite Ds elements internally contain rearranged Ac
and unrelated sequences.
Transposition
1. Insertion results in short target site duplication (“footprint”).
This suggests that the mechanism of integration involves
staggered cut (~8 bp) at the target site followed by strand
synthesis.
2. Excision is imprecise and associated with nucleotide addition,
deletion or inversion at their junction
Requirements for transposition
•
•
•
Terminal inverted repeats (TIR) are essential for transposition.
In addition to IR, ~240 bp sub-terminal nucleotides (both at 5’
and 3’ ends) of Ac are essential for transposition. At either ends
of Ac internal deletions extending farther towards the termini
results in gradual reduction in transposition. The element is
immobilized when 116 bp or less at 5’ end and 102 or less at 3’
end are retained.
Transposase (TPase)
Characteristics of Transposition
1. Ac transposes by nonreplicative mechanism. It is physically
excised from the donor site and re-inserted into the new position.
2. Ac transposes primarily, if not exclusively, during or shortly after
replication.
2. Ac transposes from only one of the two daughter chromosomes.
3. Ac insertion may occur in both replicated or unreplicated target
site.
4. Ac transposes preferentially to physically linked target site.
a
b
Ac
Ac
Ac
a
Ac
Ac
b
Ac
Ac
Ac 5’- and 3’- ends in direct orientation induce chromosome breakage
One of the discoveries of McClintock was a phenomenon she
called “breakage-fusion-bridge cycle”. Chromosome breakage
occurred frequently at genetic locus, which she therefore called
Dissociation (Ds). She later found that Ds locus could change its
position in the genome, and that transposition of Ds and
chromosome breakage required the presence of another locus,
she designated Activator (Ac).
Later other scientists elucidated that chromosome breakage is a
consequence of aberrant transposition attempts between two
different element ends in direct orientation.
L
R
L
R
McClintock’s Ds locus
Rarely transposes, often causes chromosomal breakage
Strand selectivity model
L
R
L
R
L
After replication of two directly oriented left and right ends,
one end on each daughter chromatid becomes incompetent.
After DNA cleavage at the termini of the two competent
ends, religation of the two ends leads to dicentric
chromosome.
Ac-TPase expression
1.
2.
3.
Most of the Ac DNA sequence codes for for Ac-TPase.
Ac promoter lacks a CAAT and TATA box and therefore is reminiscent
mammalian housekeeping gene promoters.
Like these, Ac promoter is weak and constitutively active.
Ac-TPase structure
•
•
•
•
•
•
It has 807 amino acids.
The N-terminal contains 3 NLS.
Between first and second NLS is TPase catalytic domain.
Overlapping with NLS 2 and 3 is a DNA binding domain.
TPase binds to sub-terminal, repetitive sequence motifs.
DNA binding domain of TPase is methylation sensitive.
Ac/ Ds can transpose in heterologous plant systems
1. tobacco.
2. Rice
3. Wheat
4. Arabidopsis
5. Tomato
6. petunia
En/Spm transposable element system of maize
Enhancer (En)/Suppressor-mutator (Spm)
13 bp perfect terminal inverted repeat
P
ATG
ATG
8.3 kb
tnpD
tnpA
Makes a 3-bp footprints (target site duplication)
En/Spm Transposable elements
cis Determinants for Excision: Approx. 180 bp at the 5’ end and 300 bp of 3’
end represent cis determinants. Contained in these regions are reiterations
of a 12-bp sequence motif that is recognized by TNPA protein with 6 motifs
present at 5’ end and 8 at 3’ end.
Trans-factors: TNPA and TNPD (transposase complex)
TNPA brings the two ends together and
causes DNA bending, TNPD cleaves
TNPA
TNPD
The “suppressor” function of En/Spm Transposable elements
En/Spm have a unique feature: they act as suppressor of gene function
The non-autonomous derivative of En/Spm (dSpm) when inserted into a
gene causes reduced gene expression of that gene instead of knocking it
out. The residual gene activity is due to the spicing of dSpm from pre-mRNA.
However, if trans-factors TNPA is present then gene activity is knocked out
i.e. pre-mRNA is not formed. TNPA binding with dSpm probably causes
steric hindrance for RNA polymerase.
Mutator Transposable element system of maize
Mutator (Mu) trait was first identified by Robertson (1978) as a heritable high
forward-mutation rate exhibited by maize lines. Many of these de novo
mutations exhibited somatic instability, primarily apparent reversion to wildtype. This phenomenon was found to be associated with Mu transposable
elements.
Autonomous MuDR element (4942-bp): 200-bp TIR, create 9-bp target site
duplication.
TIR
P
2.8-kb RNA (823 aa peptide)
1-kb RNA
(207 aa peptide)
Both proteins tightly bind to TIRs
P
TIR
Several Mu elements (subfamilies) exist that contain variable internal sequence.
These are non-autonomous derivatives of MuDR.
Applications:
1. Mu elements are known to transpose to any locus, especially genes, therefore it
is very useful for creating tagged mutations.
2. Mutator’s frequent transposition activity (even to unlinked locus) is reminiscent of
P element system of Drosophila. In Drosophila, P elements have been used as
vectors to increase the efficiency of transgene integration in the injected oocytes.
Therefore, Mu is an attractive system for increasing transformation efficiency of
maize. The strategy would involve introducing gene-of-interest in a Mu element
and then injecting that into maize cells containing MuDR (MuDR based cloning in
impossible because MuDR is unstable in E. coli). However, no report has so far
been published that describes the success for this strategy.

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