Genetic Recombination

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Genetic Recombination
Definition The breakage and joining of DNA into
new combinations

• Plays a major role in repair of damaged DNA and
  mutagenesis

• Critical for several mechanisms of phase and
  antigenic variation

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• 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)

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Homologous Recombination

• Step One
• Formation of complementary base pairing between
  two ds DNA molecules

- Sequences must be the same or very similar - 23
base pair minimum
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Step two Branch migration to extend the region
of base-pairing (the heteroduplex)

• ATP-hydrolyzing proteins (Ruv proteins) break and
  re-form H bonds
• allow migration to go faster

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Branch extension can increase the chance of gene
conversion via increasing the chances of
including mismatches in the heteroduplex region
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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
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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/holli
  day3D.html for strand resolution

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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
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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
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Proteins involved in DNA recombination (the E.
coli paradigm)
Mutation Phenotype
RecA RecBC RecD RecF RecJ RecO RecR RecQ RecN
RecG RuvA RuvB RuvC PriA PriB PriC DnaT
Recombination deficient Reduced
recombination Rec ? independent
Reduced plasmid recombination
Reduced recombination in RecBC- as above as
above as above
Reduced recombination in RecBC- Reduced
recombination in RuvA-B-C-
Reduced recombination in RecG- as
above as above
Reduced recombination as above as
above as above
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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

• Upon cessation of exonuclease activity,
• the undegraded 3 end pairs with
• homologous sequences on another
• DNA molecule

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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

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RecA protein-dsDNA complex imaged by atomic force
microscopy www-mic.ucdavis.edu
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Proteins involved in DNA recombination (the E.
coli paradigm) (cont)
Mutation Phenotype
RecA RecBC RecD RecF RecJ RecO RecR RecQ RecN
RecG RuvA RuvB RuvC PriA PriB PriC DnaT
Recombination deficient Reduced
recombination Rec ? independent
Reduced plasmid recombination
Reduced recombination in RecBC- as above as
above as above
Reduced recombination in RecBC- Reduced
recombination in RuvA-B-C-
Reduced recombination in RecG- as
above as above
Reduced recombination as above as
above as above
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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

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RuvB
RuvB

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
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a
b
a
AmpR
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b
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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 -
integrases recognize and promote recombination
between two sequences
of DNA - 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 are
present in reverse orientation to each
other
intermolecular
intramolecular
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Lytic/Lysogenic Developmental Switch
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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

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integrase
excisase
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Phage integration and excision (cont)

• 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
• 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

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Lysogenic state
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Examples of site-specific recombination
(cont) 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
Inverted repeats