Genetic Recombination
Definition: The breakage and joining of DNA into new combinations
Purposes
• Promotes genetic diversity within a species
- within a chromosome
causes inversions, deletions, duplications
- horizontal exchange
introduces new sequences (information)
• Plays a major role in repair of damaged DNA and mutagenesis
• Critical for several mechanisms of phase and antigenic variation
In the lab:
introduce foreign DNA or mutations into bacteria
map the distance between mutations
Types:
• Homologous recombination (or general recombination)
• basic steps
• current models
• proteins that play a role
• practical applications
• Nonhomologous recombination (site-specific)
•Basic steps
• general categories of proteins used
• examples – phage integration, flagellin phase variation
• Illegitimate recombination (transposition)
Homologous Recombination
Step One
• Formation of complementary base pairing between two ds DNA molecules
CACATGATACGTCCGATCACATTTGTTGTTCATAT
GTGTACTATGCAGGCTAGTGTAAACAACAAGTATA
GTGTACTATGCAGGCTAGTGTAAACAACAAGTATA
CACATGATACGTCCGATCACATTTGTTGTTCATAT
- Sequences must be the same or very similar
- 23 base pair minimum
• Results in the creation of a synapse [synapse is point where DNA strands are held
together by complementary base-pairing (H bonds)]
CACATGATACGTCCGATCACATTTGTTGTTCATAT
GTGTACTATGCAGGCTAGTGTAAA C
GTATA
A
A
A
A
C
GTATA
GTGTACTATGCAGGCTAGTGTAAA
CACATGATACGTCCGATCACATTTGTTGTTCATAT
Synapse
Step two
Branch migration to extend the region of base-pairing (the heteroduplex)
CACATGATACGTCCGATCACATTTGTTGTTCATAT
GTGTACTATGCAGGCTAGTGTAAA C
GTATA
A A
AA
C G
GTATA
GTGTACTATGCAGGCTAGTGTAAA
CACATGATACGTCCGATCACATTTGTTGTTCATAT
CACATGATACGTCCGATCACATTTGTTGTTCATAT
GTGTA
GGCTAGTGTAAACAACAAGTATA
C
A
T
A
C
T G
GT
Branch migration
A
T CA
C
GGCTAGTGTAAACAACAAGTATA
GTGTA
CACATGATACGTCCGATCACATTTGTTGTTCATAT
-ATP-hydrolyzing proteins (Ruv proteins) break and re-form H bonds
allow migration to go faster
Branch extension can increase the chance of gene conversion via increasing the
chances of including mismatches in the heteroduplex region
CACATGATACGTCCGATCACATTTGTTGTTCATAT
GTGTACTATGCAGGCTAGTGTAAA C
GTATA
A A
AA
C G
GTATA
GTGTACTATGCAGGCTGGTGTAAA
CACATGATACGTCCGACCACATTTGTTGTTCATAT
CACATGAT ACGT CCGATCACATTTGTTGTTCATAT
GTGTA
GGCTGGTGTAAACAACAAGTATA
C
A
T
A
C
T G
GT
Branch migration
A
T CA
C
GGCTAGTGTAAACAACAAGTATA
GTGTA
CACATGAT AC GT CCGACCACATTTGTTGTTCATAT
Step three
Resolution of the heteroduplex
- isomerization of the duplex due to strands uncrossing and re-crossing
- results in different outcomes upon resolution
- 50% chance of each isomer being resolved
Models of Homologous Recombination (I)
Holliday double-strand invasion
Fig. 10.1 of textbook
• Initiated by two single-stranded breaks made simultaneously and exactly
in the same place in the DNA molecules to be recombined
• Free ends of the two broken strands cross over each other, pairing with its
complementary sequence in the other DNA molecule to form heteroduplex.
See http://engels.genetics.wisc.edu/Holliday/holliday3D.html for strand resolution
Models of Homologous Recombination (II)
Single-strand Invasion
• Single strand break in one molecule
• Free ss end invades other DNA molecule
• Gap on cut DNA is filled in by
DNA polymerase
• Displaced strand on other DNA molecule
is degraded
• Two ends are joined
• Initially, heteroduplex is only on
one strand; branch migration causes
another heteroduplex on other molecule
Fig. 10.3 of textbook
Models of Homologous Recombination (III)
Double strand break-repair
• A double stranded break occurs in one
molecule; and exonuclease digests the
5’ ends of each break, leaving a gap
• One of the 3’ tails invades unbroken
molecule; pairs with complementary
sequence
• DNA polymerase extends the tail until
it can be joined with 5’ end
• Displaced strand used as template to
replace gap on other molecule
• Two Holliday junctions form (may
produce recombinant flanking DNA
depending how they are resolved)
Fig. 10.4 of textbook
Proteins involved in DNA recombination (the E. coli paradigm)
Mutation
Phenotype
RecA
Recombination deficient
RecBC
Reduced recombination
RecD
Rec+
RecF
Reduced plasmid recombination
RecJ
RecO
RecR
RecQ
Reduced recombination in RecBCas above
as above
as above
 independent
RecN
RecG
Reduced recombination in RecBCReduced recombination in RuvA-B-C-
RuvA
RuvB
RuvC
Reduced recombination in RecGas above
as above
PriA
PriB
PriC
DnaT
Reduced recombination
as above
as above
as above
+
or
Donor
DNA
Mutant bank
(i.e. of E. coli)
Screen for inability
to acquire a
selectable marker
RecBCD exonuclease: opens strands
• RecBCD binds to DNA at one end or at
a ds breakage point
• Moves along the DNA, creating a loop
and degrading the strand with a free
3’ end via its exonuclease activity
• Exonuclease activity is inhibited
upon passing a Chi site of orientation
Chi (Χ) site:
• 8 base pair sequence without
symmetry (5’GCTGGTCC)
• Greatly stimulates ability of RecBCD
to catalyze recombination
• Upon cessation of exonuclease activity,
the undegraded 3’ end pairs with
homologous sequences on another
DNA molecule
RecA: needed to form triple helix
• RecA binds to free strand to form an
extended helical structure.
• Resultant DNA-RecA helix forms a
triple-stranded helix with ds DNA
that has a homologous region
• one of the strands in the ds helix is
displaced (D loop)
• displaced strand binds to original
complementary strand of the invasive
strand to create Holliday junction
RecA protein-dsDNA complex imaged by atomic force microscopy
www-mic.ucdavis.edu
Proteins involved in DNA recombination (the E. coli paradigm) (con’t)
Mutation
Phenotype
RecA
Recombination deficient
RecBC
Reduced recombination
RecD
Rec+  independent
RecF
Reduced plasmid recombination
RecJ
RecO
RecR
RecQ
Reduced recombination in RecBCas above
as above
as above
RecN
RecG
Reduced recombination in RecBCReduced recombination in RuvA-B-C-
RuvA
RuvB
RuvC
Reduced recombination in RecGas above
as above
PriA
PriB
PriC
DnaT
Reduced recombination
as above
as above
as above
RecF pathway
• important for DNA repair
(i.e. UV light)
• detectable as reduced
recombination in RecBCbackground
Important after heteroduplex
formation is initiated
-branch migration
- resolution of heterduplex
Efficient branch migration requires RuvA and RuvB
• RuvA specifically binds Holliday junctions
- resultant structure better able to undergo branch migration and resolution
• RuvB is a helicase
- forms a hexameric ring around the DNA strand
- DNA is pumped through the ring using ATP cleavage to drive the pump
- the synapse is thus forced to migrate
RuvB
RuvA
RuvC resolves (cuts) the Holliday junction
• Ruv C is a specialized endonuclease
an X-phile – cuts crossed DNA strands
always cuts at two T’s
RuvB
RuvB
RuvA
A simple model of a RuvA/RuvB/DNA complex extrapolating from the above model and
in agreement with the electron microscopy results of Parsons et al. (Nature 374, 375
(1995)). RuvA binds the Holliday junction at the central crossover point and targets two
RuvB hexamers onto opposite arms of the DNA where they encircle the DNA duplexes
and facilitate branch migration in concert with RuvA in an ATP dependent manner.
For animation, see
http://www.sdsc.edu/journals/mbb/ruva.html
a
b
AmpR
b’
a a’
EmR
b
Or
AmpR
EmR
a
5’ end
of gene
a
a
a
Single cross-over results in one truncated copy
and one intact copy of the gene
a
internal
fragment
a
Single cross-over results in an interrupted gene
a
a
a
b
b
a
Single cross-over outcome when using
one end of the gene
P1
P1
a
b
P2
a
a
b
a
Useful for introducing a promoter-reporter gene fusion
without disrupting the gene’s function.
Nonhomologous (Site-specific) Recombination
• Occurs at specific or highly preferred target and donor DNA sequences
• Requires special proteins that recognize specific sequences and
catalyze the molecular events required for strand exchange
• Relatively rare compared to homologous recombination
Site-specific recombinases include:
intermolecular
- integrases recognize and promote recombination between
two sequences of DNA
Example:
phage integrases
intramolecular
- resolvases resolve co-integrates by pairing sequences within sites
that are present in direct orientation to each other
(example - transposon resolvases)
- invertases pair sequences within sites that
Example:
are present in reverse
Salmonella flagellin
orientation to each other
phase variation
Lytic/Lysogenic Developmental Switch
Examples of site-specific recombination
1) Phage integration and excision
• Integration of circular phage
DNA into the host DNA to
form a prophage occurs via
the action of phage Int
enzymes (integrases).
• Usually highly specific and
occurs at only one or a few
integration sites on the
chromosome
• Excision utilizes both the
integrase and an excisase,
which act at the hybrid
integration sites that flank the
prophage
integrase
excisase
Phage integration and excision (con’t)
•
Lysogenization by lambda phage:
Site-specific recombination between the attP site on phage and
the attB site on bacterial chromosome
attP and attB are dissimilar except for 15 bp core sequence
GCTTTTTTATACTAA
GCTTTTTTATACTAA
The lambda Int protein is an integrase that promotes site-specific
recombination between 7 internal bases of the core sequence
•
Excision is via production on integrase (Int) and excisase (Xis), which promote
recombination of the hybrid attP/B and attB/P molecules in the chromosome
Lysogenic
state
Examples of site-specific recombination (con’t)
2) Phase variation of Salmonella flagellin genes
•
Reversible, high frequency (10-4) inversion of DNA sequence that carries
the promoter for one flagellin structural gene and for a repressor of
a second flagellin gene
•
Occurs by virtue of a DNA invertase called Hin
•
Promotes site-specific recombination between two closely linked
sites of DNA
P
hin
Inverted repeats
H2 flagellin
Repressor
H1 flagellin