Download DNA Recombination

DNA Recombination
• Involves the physical exchange of DNA sequences from one
molecule DNA to another molecule.
• Importance of DNA recombination:
- providing genetic variation (Genetic variation is crucial to allow
organisms to evolve in response to a changing environment).
- replacing damaged DNA with an undamaged strand.
- regulation of gene expression.
• Major aspect of homologous recombination
- breakage of DNA
- joining of DNA
Key steps of homologous recombination
• Alignment of two homologous DNA molecules.
• Introduction of breaks in the DNA.
- These breaks are further processed to generate regions of singlestranded DNA.
• Formation of initial regions of base pairing between recombining
DNA molecules.
- Single-stranded region of DNA originating from one parental molecule
pairs with its complementary strand in the homologous duplex DNA
molecule (strand invasion, generating heteroduplex DNA).
• Then, two DNA molecules become connected by crossing DNA
strands generating Holliday junction.
- When the junction moves, base pairs are broken in the parental DNA
molecules while identical base pairs are formed in the recombination
intermediate, branch migration.
• Clevage of the Holliday junction.
- Two pairs of DNA strands in the Holliday junction are cut during
resolution.
• Special endonuclease that simultaneously
cut both strands of the double helix, creating a
complete break in the DNA molecule.
• The 5’ ends at the break are chewed back
by an exonuclease, creating a protruding
single-stranded 3’ ends.
• These single stranded then search for a
homologous DNA helix with which to pair,
leading to the formation of a Holliday junction.
• Strand invasion generates a Holliday
junction that can move along the DNA by
branch migration (increased the length of DNA
exchanged).
• If the two DNA molecules are not identical,
branch migration through these regions of
sequence difference generates DNA duplexes
carrying one or few sequence mismatches.
• Recombination is completed when Holliday
junction is resolved (two recombining DNA
molecules are separated).
• Resolution of Holliday junctions occurs in one of the two ways and
therefore give rise to two distinct classes of DNA products.
• Horizontal resolution: Patch products or noncrossover products (No
reassortment of flanking genes).
• Vertical resolution: Splice products or crossover products (Reassortment
of flanking genes).
Genetic
Recombination
Conservative
site-specific
recombination
Transpositional
recombination
• Conservative site-specific recombination (CSSR)
- recombination between two defined sequence elements.
• Transpositional recombination (transposition)
- recombination between specific sequences and nonspecific DNA
sites.
• Recombinases
- recognize specific sequences where recombination will occur
within a DNA molecule
- bring specific sites together to form a protein-DNA complex
bridging the DNA sites.
- catalyzes the cleavage and rejoining of the DNA molecules either
to invert a DNA segment or to move a segment to a new site.
Conservative Site-Specific Recombination
• Key feature: segment of DNA that will be moved carries specific
short sequence elements, recombination sites (~20bp), where DNA
exchange occurs.
• Recombination sites carry:
i) sequences specifically bound by the recombinases.
ii) sequences where DNA cleavage and rejoining occur.
• CSSR generate three different types of DNA rearrangements:
i) insertion of a segment of DNA into a specific site.
ii) deletion of a DNA segment.
iii) inversion of a DNA segment.
• Example: Integration of the bacteriophage λ genome into bacterial
chromosome.
Three types of CSSR recombination
which is depending on the organization of the recombinase recognition sites on
the DNA molecule or molecules that participate in recombination.
Structures involved in CSSR
• Each recombination sites is organized
as a pair of recombinase recognition
sequences and positioned symmetrically.
• The recognition sequences flank a
central short asymmetric sequence,
known as crossover region, where DNA
cleavage and rejoining occurs.
• The subunits of the recombinase bind
these recognition sites and recombination
occurs.
• Two families of conservative site-specific recombinases:
i) serine recombinases
ii) tyrosine recombinases
• Key feature: covalent-DNA intermediate is generated when the DNA
is cleaved
• Serine recombinases:
- Side chain of the active-site serine residue attacks and then
becomes joined to the
- 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.
• Tyrosine recombinases:
- Side chain of active-site tyrosine residue attacks and then
becomes joined to the DNA.
- 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.
Transposition
• Moves certain genetic elements from one DNA site to another.
• Mobile genetic elements are called transposable elements or
transposons.
• Movement occurs through recombination between the DNA
sequences at the very ends of the transposable element and a
sequence in the DNA of the host cell.
• Transposons show little sequence selectivity in their choice of
insertion sites. Therefore, transposons can insert:
i) within genes → disrupting gene function.
ii) regulatory sequences of a gene → alter gene expression.
• Transposons are important cause of mutations leading to genetic
disease in humans.
• The ability of transposons to insert so promiscuously in DNA led to
their modification and use as mutagens and DNA-delivery vectors in
experimental biology.
Trsnposition of a mobile genetic element to a new site in the host
DNA
• Involves excision of the transposon from the old DNA location and
insertion to a new site.
• Or, one copy of the transposon stays at the old location and another
copy is inserted into the new DNA site.
Three classes of Transposable Elements
• DNA transposons
• Viral-like retrotransposons
- long terminal repeat (LTR) retrotransposons
• Non-viral retrotransposons
- poly A retrotransposons.
DNA Transposons
• Carry both DNA sequences that function as recombination sites and genes
encoding proteins that participate in recombination.
• Recombination sites are at two ends of the element and are organized as
inverted-repeat sequences.
• Recombinases responsible for transposition are called transposases (or
integrases).
• Autonomous transposons
- carry a pair of terminal inverted repeats and a tranposase gene.
- function independently
• Non-autonomous transposons
- carry only the terminal inverted repeats.
- require the presence transposase encoded by autonomous transposons to enable
transposition.
Viral-like Retrotransposons
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Carry long inverted terminal repeat sequences (LTRs).
Retrotransposon encode two proteins: integrase (transposase) and reverse
transcriptase.
Reverse transcriptase uses RNA template to synthesize DNA.
Non-viral retrotransposons
• Do not have the terminal inverted repeats present in the other transposon
classes.
• The two ends of the element have distinct sequences, 5’UTR and 3’UTR which
is followed by poly-A sequence.
• Carry two genes, ORF1 (RNA-binding protein) and ORF2 (reverse
transcriptase and endonuclease).
c. Non-viral retrotransposons
DNA transposition by a Cut-and-Paste Mechanism
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Transposase bind to the terminal inverted repeats at the end of the
transposon.
Transposase brings the two ends of the tranposon DNa together to
generate a stable protein-DNA complex (synaptic complex or
transpososome which is essential to coordinate DNA cleavage and joining
reactions on the two ends of the transposon’s DNA).
Transposase cleaves DNA such that transposon sequence terminates with
free 3’-OH groups at each end of the element’s DNA.
3’-OH ends of the transposon DNA attack the DNA phosphodiester bonds at
the site of new insertion.
Gap repair by DNA polymerase.
DNA transposition by a Replicative Mechanism
• Element DNA is duplicated during each round of transposition.
• Mechanism:
i) Assembly of the transposase protein on the two ends of the transposon
to generate a transpososome.
ii) DNA cleavage at the ends of the transposon DNA. Transposase
introduces a nick into DNA at each of the junctions between the transposon
sequence and the flanking host DNA.
iii) The 3’OH ends of transposon DNA are then joined to the target DNA
site by the DNA strand transfer reaction whereas 5’ ends of the transposon
sequence remain joined to the old flanking DNA.
iv) The 3’-OH end in the cleaved target DNA serves as a primer for DNA
synthesis. Replication proceeds through the transposon sequence and stops
at the second fork.
Mechanism of retroviral
integration and transposition of
virus-like retrotransposons.
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Transcription of the retrotransposon
DNA sequence into RNA by cellular
RNA polymerase.
The RNA is then reverse-transcribed
to generate double-stranded DNA
molecule, cDNA.
Integrase assembles on the ends of
the cDNA and then cleaves a few
nucleotides off the 3’ end of each
strand.
Integrase then catalyzes the insertion
of cleaved 3’ ends into a DNA target
site in the host cell genome using
DNA strand transfer reaction.
DNA repair proteins fill the gaps at the
target site generated during DNA
strand transfer to complete
recombination.
Non-retroviral retrotransposon move by
reverse splicing mechanism
• Cellular RNA polymerase initiates transcription of
and integrated LINE sequence.
• The resulting mRNA is translated to produce
products of the two encoded ORFs that then bind to
the 3’ end of their mRNA.
• The protein-mRNA complex then binds to a T-rich
site in the target DNA.
• The proteins initiates cleavage in the target DN,
leaving 3’-OH at the DNA end forming an RNA:DNA
hybrid.
•The 3’-OH end of the target DNA serves as a primer
for reverse transcription of the element RNA to
produce cDNA.
• The final steps of transposition reaction include
second-strand synthesis and DNA joining and repair
to crate a newly inserted LINE element.