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Biological Roles for Recombination
1. Generating new gene/allele combinations
(crossing over during meiosis)
2. Generating new genes (e.g., Immunoglobulin rearrangement)
3. Integration of a specific DNA element (or
4. virus)
5. DNA repair
Why Recombination?
Mutation happens - without recombination, mutation target
would increase from gene to entire chromosome
Recombination allows favorable and unfavorable mutations
to be separated
Provides a means of escape, to generate new combinations
of genes, and spreading of favorable alleles
Two Broad Categories
Homologous (or general)
Site-specific (e.g. phage genomes into bacterial chromosomes)
Homologous Recombination
Required for DNA replication, repairs accidents at
replication fork
Repairs double strand DNA (dsDNA) breaks
Occurs at meiosis (cross-overs)
Happens at “four strand” stage of meiosis, involves
two of four strands
Occurs randomly between homologous sequences
Double strand break repair (DSBR) model
Allelic and non-allelic (ectopic recombination)
• Homologous recombination
• Nonhomologous recombination.
• Homologous recombination is based on
sequence complementarity, and can be
further divided into:
•
generalized recombination
•
site- specific recombination.
Generalized recombination refers to DNA
exchange between molecules with extended
sequence homology.
For example, transformation and conjugation
between related bacterial strains.
Site-specific recombination refers to DNA
recombination between molecules that shared
limited regions of sequence homology.
For example, conjugation between two different
species.
Two models have been proposed for generalized
recombination:
a. Single stranded break model:
Recombination starts with the nick or break of
homologous strands at a single correspondent
point.
This breakage allows each free end to pair
with its complement in the other duplex).
B. The double stranded break model
DNA exchange is initiated by an endonucleaseinduced, double strand break in one molecule
called recipient duplex.
The breakage is enlarged by the endonuclease
to a gap with 3’ end free ends.
Nonhomologous recombination refers to the
process by which a DNA molecule is
incorporated
into an unrelated genome with no sequence
homology.
For exp., transposition of viral genome into
human chromosome, or transposition.
Mechanisms of generalized recombination:
Recombiantion involves breakage and rejoining
of homologous DNA molecules.
The intermediate stage during homologous
recombination where the two homologous
molecule covalent linked to each other is called
Holiday Structure.
Examples of (mostly) Homologous Recombination
Fig. 22.1
The Holliday model
R. Holliday (1964)
Two homologous duplexes are aligned
Strand exchange leads to an intermediate
with crossed strands
This branch can move: Branch migration
The branch is resolved by cleavage and
sealing
Holliday Model
- Holliday Junctions
form during
recombination
- HJs can be resolved
2 ways, only one
produces true
recombinant
molecules
patch
The recBCD
Pathway of
Homologous
Recombination
Part I: Nicking and
Exchanging
recBCD Pathway of Homologous Recomb.
Part I: Nicking and Exchanging
1.
2.
3.
4.
5.
A nick is created in one strand by recBCD at a Chi
sequence (GCTGGTGG), found every 5000 bp.
Unwinding of DNA containing Chi sequence by
recBCD allows binding of SSB and recA.
recA promotes strand invasion into homologous
DNA, displacing one strand.
The displaced strand base-pairs with the single
strand left behind on the other chromosome.
The displaced and now paired strand is nicked
(by recBCD?) to complete strand exchange.
Recombination is initiated by
double-strand breaks in DNA
Double-stranded (dsDNA) breaks
are not uncommon
Meiosis
Created by topoisomerase-like enzymes
Mitosis
Radiation
Mutagens (e.g. chemicals)
Stalled replication forks
Specialized endonucleases (eg site-specific HO endonuclease in
switching of yeast matting type (MAT) genes)
Recombination requires DNA binding
proteins
Extensively studied in model organisms, E. coli and yeast
Bacterial recombination enzymes identified by Rec - mutations
At least 25 proteins are involved in homologous recombination in E.
coli
Remember four; RecBCD and RecA
RecBCD
3 member protein complex with endonuclease and
helicase activity
essential for 99% of recombination events
occurring at double-stranded breaks in bacteria
binds double stranded break
unwinds and degrades DNA
Pauses at chi sequence
Loads RecA on 3’ ssDNA extensions
Initiation of recombination by the
RecBCD enzyme
RecA enables single stranded DNA
to invade DNA helix
RecA
Involved in SOS response; required for nearly ALL
homologous
recombination in bacteria
Single-strand DNA binding protein, DNA dependent ATPase
Multiple DNA binding sites
Initiates the exchange of DNA between two recombining
DNA double helixes
Eukaryotes have multiple homologs of bacterial RecA
(Rad51 is best studied)
Chi site Χ
Recombination hotspot
Modifies RecBCD enzymatic activity
5’ GCTGGTGG 3’
1009 chi (Χ) sites in E. coli genome
Χ homologs in other bacteria
Targeted gene disruption by
homologous recombination
Lodish et al. Molecular Cell Biology
Gene Conversion
A special type of homologous recombination
Non-reciprocal transfer of genetic material from a ‘donor’
sequence to a highly homologous ‘acceptor’ sequence
Initiated by double strand DNA (dsDNA) breaks
5’ > 3’ exonucleases
3’ ssDNA tail strand invasion (RAD51 and others)
Outcome: portion of ‘donor’ sequence copied to ‘acceptor’
and original ‘donor’ copy unchanged
gene
conversion
donor
acceptor
recBCD Pathway of Homologous Recom.
Part II: Branch Migration and Resolution
1. Nicks are sealed  Holliday Junction
2. Branch migration (ruvA + ruvB)
3. Resolution of Holliday Junction (ruvC)
RecBCD : A Complex Enzyme
• RecBCD has:
1. Endonuclease subunits (recBC) that cut
one DNA strand close to Chi sequence.
2. DNA helicase activity (recD subunit) and
a DNA-dependent ATPase activity
– unwinds DNA to generate the 3’ SS tails
RecA
• 38 kDa protein that polymerizes onto SS DNA 5’-3’
• Catalyzes strand exchange, also an ATPase
• Also binds DS DNA, but not as strongly as SS
RecA binds preferentially
to SS DNA and will
catalyze invasion of a DS
DNA molecule by a SS
homologue.
Important for many types
of homologous
recombination, such as
during meoisis (in yeast).
RecA Function Dissected
• 3 steps of strand exchange:
1. Pre-synapsis: recA coats single-stranded
DNA (accelerated by SSB, so get more
relaxed structure).
2. Synapsis: alignment of complementary
sequences in SS and DS DNA (paranemic
or side-by-side structure).
3. Post-synapsis or strand-exchange: SS DNA
replaces the same strand in the duplex to
form a new DS DNA (requires ATP
hydrolysis).
Meiotic
Recomb. in
Yeast
- is initiated by a
double-strand
break (DSB)
Repair of double-strand breaks (DSBs)
in non-dividing or mitotic cells
DSBs probably most severe form of DNA
damage, can cause loss of genes or even
cell death (apoptosis)
DSBs caused by:
- ionizing radiation
- certain chemicals
- some enzymes (topoisomerases,
endonucleases)
- torsional stress
2 general ways to repair DSBs:
1.
Homologous recombination (HR) - repair of broken
DNA using the intact homologue, very similar to
meiotic recombination. Very accurate.
2.
Non-homologous end joining (NHEJ) - ligating nonhomologous ends. Prone to errors, ends can be
damaged before religation (genetic material lost) or
get translocations. (Mechanism in Fig 20.38)
Usage: NHEJ >> HR in plants and animals
DNA Recompilation applications
What the is Recombinant DNA?
Recombinant DNA is what you get when you combine
DNA from two different sources.
For example:
Mouse + Human DNA
Human + Bacterial DNA
Viral + Bacterial DNA
Human + (other) Human DNA
Why Make Recombinant DNA?
Recombinant DNA Technology May Allow Us
To:
• Cure or treat disease
• Genetically modify our foods to increase
flavor, yield, nutritional value or shelf-life
• Better understand human genetics
• Clone cells or organs
Molecular Biology’s Best Friends:
Bacteria
Why use bacteria?
• They’re relatively simple organisms.
• They reproduce very quickly and asexually
(this means that the “daughter” cells will
contain the exact same DNA as the “parent”
cell).
• It’s pretty easy to get DNA back into the
bacteria after you’ve changed it.
• We can mess around with their DNA and kill a
lot of them during our experiments and
nobody gets mad.
Insulin for Diabetics: The New Way
Step 1:
Isolate (find) the human gene responsible for
producing insulin
and decide where you want to put it.
In this case, we decide to put our human DNA
into the plasmid of E. coli, a very
common bacterium.
Step 2:
Get the bacterial (plasmid) DNA out of the E. coli.
We do this by basically exploding them.
Step 3:
Cut your human DNA and bacterial DNA with the
same restriction enzyme.
Step 4:
Mix the cut human DNA, which contains the insulin
gene, with the cut bacterial DNA.
They’ll stick together because they were cut with
the same restriction enzyme.
Step 5:
Get your new recombinant plasmid back
into the bacteria.
This is easy because bacteria will take in
DNA that’s floating around near them. We
call this “transformation”.
Step 6
• Find the bacteria that have taken up your
recombinant plasmid amongst the riff-raff in the
petri dish.
Now your E. coli will use its new DNA to make
human insulin!
Because they reproduce so quickly, you’ll
soon have thousands, millions, or billions of
human insulin making machines.
By filtering out the bacteria after they’ve
made insulin, you’ve got clean human insulin
that can be packaged and given to diabetic
patients.