Genetic Recombination

Definition: The breakage and joining of DNA into new combinations

• Critical for several mechanisms of phase and antigenic variation

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

Purposes

• Promotes genetic diversity within a species- within a chromosome causes inversions, deletions, duplications

- horizontal exchange introduces new sequences (information)

In the lab:

map the distance between mutations

introduce foreign DNA or mutations into bacteria

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

CACATGATACGTCCGATCACATTTGTTGTTCATATGTGTACTATGCAGGCTAGTGTAAACAACAAGTATA

GTGTACTATGCAGGCTAGTGTAAACAACAAGTATACACATGATACGTCCGATCACATTTGTTGTTCATAT

- 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 GTGTACTATGCAGGCTAGTGTAAAC

Synapse

C

CACATGATACGTCCGATCACATTTGTTGTTCATATGTGTACTATGCAGGCTAGTGTAAA

A AGTATA

AA

GTATA

Step two Branch migration to extend the region of base-pairing (the heteroduplex)

CACATGATACGTCCGATCACATTTGTTGTTCATAT GTGTA GGCTAGTGTAAACAACAAGTATAC

TA

TGT

GCA

C

GGCTAGTGTAAACAACAAGTATACACATGATACGTCCGATCACATTTGTTGTTCATAT

AGTGTA

CTA

Branch migration

CACATGATACGTCCGATCACATTTGTTGTTCATAT GTGTACTATGCAGGCTAGTGTAAAC

C

CACATGATACGTCCGATCACATTTGTTGTTCATATGTGTACTATGCAGGCTAGTGTAAA

AA

GTATAA

AG

GTATA

-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

CACATGAT ACGT CCGATCACATTTGTTGTTCATAT GTGTA GGCTGGTGTAAACAACAAGTATAC

TA

TGT

GCA

C

GGCTAGTGTAAACAACAAGTATACACATGAT AC GT CCGACCACATTTGTTGTTCATAT

AGTGTA

CTA

Branch migration

CACATGATACGTCCGATCACATTTGTTGTTCATAT GTGTACTATGCAGGCTAGTGTAAAC

C

CACATGATACGTCCGACCACATTTGTTGTTCATATGTGTACTATGCAGGCTGGTGTAAA

AA

GTATAA

AG

GTATA

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)

Fig. 10.1 of textbook

Holliday double-strand invasion

• 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

Fig. 10.3 of textbook

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

Models of Homologous Recombination (III)

Double strand break-repair

Fig. 10.4 of textbook

• 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)

Proteins involved in DNA recombination (the E. coli paradigm)

RecA

RecBC

RecD

RecF

RecJRecORecRRecQ

RecNRecG

RuvARuvBRuvC

PriAPriBPriCDnaT

Recombination deficient

Reduced recombination

Rec+ independent

Reduced plasmid recombination

Reduced recombination in RecBC-

as aboveas aboveas above

Reduced recombination in RecBC-

Reduced recombination in RuvA-B-C-

Reduced recombination in RecG-

as above as above

Reduced recombination as above

as aboveas above

Mutation Phenotype

Screen for inability to acquire a

selectable marker

Mutant bank (i.e. of E. coli)

DonorDNA

or

+

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

Chi (Χ) site: • 8 base pair sequence without symmetry (5’GCTGGTCC)

• Greatly stimulates ability of RecBCD to catalyze recombination

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)

RecA

RecBC

RecD

RecF

RecJRecORecRRecQ

RecNRecG

RuvARuvBRuvC

PriAPriBPriCDnaT

Recombination deficient

Reduced recombination

Rec+ independent

Reduced plasmid recombination

Reduced recombination in RecBC-

as aboveas aboveas above

Reduced recombination in RecBC-

Reduced recombination in RuvA-B-C-

Reduced recombination in RecG-

as above as above

Reduced recombination as above

as aboveas above

RecF pathway• important for DNA repair (i.e. UV light)• detectable as reduced recombination in RecBC- background

Important after heteroduplex formation is initiated-branch migration- resolution of heterduplex

Mutation Phenotype

Efficient branch migration requires RuvA and RuvB

RuvB RuvA

• 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

RuvC resolves (cuts) the Holliday junction

• Ruv C is a specialized endonuclease an X-phile – cuts crossed DNA strandsalways cuts at two T’s

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

RuvA

RuvBRuvB

a

a

a

a

a

5’ end of gene

a

internalfragment

a a

Single cross-over results in one truncated copy and one intact copy of the gene

a

Single cross-over results in an interrupted gene

b

EmR

b’

b

AmpR

EmR

a’a b’

OrAmpRa’

a a b

a

a

a

a

Single cross-over outcome when using one end of the gene

b

a

P 2

a a ba

P1 P1 P2

Useful for introducing a promoter-reporter gene fusion without disrupting the gene’s function.

b

Nonhomologous (Site-specific) Recombination

• Occurs at specific or highly preferred target and donor DNA sequences

• 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

intramolecular

intermolecular

• Requires special proteins that recognize specific sequences and catalyze the molecular events required for strand exchange

Example: phage integrases

Example: Salmonella flagellin

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)

• 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

• 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

GCTTTTTTATACTAA

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

H2 flagellin Repressorhin

H1 flagellin

P

Inverted repeats