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Lecture 9 Site Specific Recombination and Transposition
Quiz 5 due today at 4 PM
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Examples of site specific recombination and transposition
Different than homologous recombination
“jumping gene”
Conservative site specific recombination (CSSR)
and transposition
• Responsible for important DNA rearrangements
CSSR = recombination between 2 defined sites
CSSR can be used to control gene expression.
DNA inversion can allow an alternative gene to be expressed.
Transposition = recombination between specific sequences
and non-specific DNA sites
Transposition is a (the) major source of spontaneous mutation.
Nearly half the human genome is transposon-derived sequences.
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Certain viruses use site specific recombination
to integrate into the host chromosome
Example: lambda phage
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3 Types of conservative site specific recombination (CSSR)
2 sites on different DNA molecules
Direct repeats
2 sites on the same DNA molecule
Direct repeats
Inverted repeats
A, B, X, and Y denote specific genes.
The recombinase recognition sequences are dark orange and blue.
Gray regions with white arrows are the crossover regions.
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Structures involved in CSSR
Example of an insertion
(orange DNA is usually circular)
Recombinase recognition
sequences are symmetric
Crossover region is assymetric
4 subunits of recombinase bind
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2 types of recombinases
Serine recombinase and tyrosine recombinase
Both use a covalent protein-DNA intermediate
Conservative in CSSR refers to energy; No ATP needed
Leaves free 3’-OH to
bind another DNA strand
Recombinase
binds 5’- phosphate
Synaptic complex
Links DNA to
3’-OH on new
strand
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Mechanism of
serine recombinase
Makes double stranded break
that is staggered by 2 bases
All 4 strands cleaved before
strand exchange
5’- phospho-serine and
free 3’-OH formed.
R2 segment recombines with
R3 segment
R4 segment recombines with
R1 segment
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Mechanism of
tyrosine recombinase
Break and rejoin one DNA
strand at a time. R1 and R3
break and rejoin segments first.
3’- phospho-tyrosine and free
5’-OH formed.
A Holliday junction is formed.
R2 and R4 segments then
recombine using the same
mechanism to resolve junction.
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Phage P1 Cre recombinase
is a tyrosine recombinase
sometimes used in genetic engineering
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Homologous recombination during
DNA replication of circular chromosomes
can generate circular multimers.
CSSR is needed to resolve these multimers
back into the monomers.
These recombinases are called resolvases.
XER tyrosine recombinase (resolvase)
is an example from bacteria
Animation
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Transposable elements or transposons
Non-replicative (cut & paste)
Replicative (copy & paste)
Little selectivity in site selection. Can insert in the middle of genes.
Less than 2% of human transposons code for proteins.
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Occurrence of transposons (green) in genomes
Some organisms (yeast, fruitflies, and E. Coli) contain few transposons.
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3 classes of transposons
(LTR retrotransposons)
Non-viral
retrotransposons
(Long terminal repeat)
(Reverse transcriptase)
RNA binding
RT & endonuclease/
enzyme
Rnase H
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DNA transposons: Cut &
paste mechanism of transposition
(transpososome)
Old host site is gray
New host site is blue
Double strand break at
old site must be repaired
Staggered cleavage of
new target site DNA
New DNA synthesis gives
rise to target site duplication
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DNA strand
transfer
3’-OH of transposon attacks
phosphate in DNA to leave a
new 3’-OH for polymerase
to add onto for the gap to be
filled. Then ligase seals nick.
Transposase only cleaves one strand. 3 ways to cleave
nontransferred strand (5’-end) before strand transfer
3’-OH on trnspsn
3’-OH on host DNA
A different endonuclease
(TnsA)
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DNA transposons: Replicative
“copy & paste” transposition mechanism
Transposase nicks DNA to create
the 3’-OH at each end for strand transfer
(same as in cut & paste)
3’-OH on each end cleaves
and binds target DNA
DNA replication machinery then uses
each 3’-OH on target DNA as a primer
and copies the transposon
This leaves a large circular DNA
molecule containing 2 copies of
the transposon
Animation
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Virus-like retrotransposons
use an RNA intermediate Integrated virus
but otherwise use the same
mechanism as in DNA transposition
Virus uses an integrase related to DNA transposases.
Integrase cleaves 2 nucleotides from the
3’-end, which is then used for DNA strand transfer.
Viruses need to use a special mechanism
to regenerate the ends of the LTRs
so they can be recognized by integrase.
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Target-site-primed reverse transcription
of Poly-A (non-LTR) retrotransposons
Human transposon LINEs (long interspersed
nuclear elements) use this mechanism.
LINE DNA transcribed. mRNA exported from nucleus.
ORF1 and ORF2 proteins are translated from mRNA,
remain attached to 3’-end of transcript, and
transport mRNA back into the nucleus where it
associates with T- rich DNA by use of the poly-A tail
To form a DNA:RNA hybrid.
ORF2 (RT and endonuclease) nicks DNA generating
a 3’-OH for reverse transcription of RNA back into
DNA that integrates into host DNA. DNA joining and
repair establish the newly produced LINE element.
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Genetic organization of a LINE and SINE
20 % of the human genome is LINE elements (example L1).
LINEs donate the proteins to replicate SINEs (100-400 bp
sequences that make up 13 % of human genome). The Alu
sequence is a SINE. Pseudogenes can be made by LINE proteins
mistakingly binding and reverse transcribing normal mRNA.
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