Picture taken in 1929 of Emerson’s corn cytogenetics class

Picture taken in 1929 of Emerson’s corn cytogenetics class at Cornell University - Beadle is a graduate student shown

here with the dog.

CHAPTER 29

The Molecular Mechanism of Recombination

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Sections 29.2 and 29.3 pages 953 to 967

Yes, you will see phage experiments AGAIN in thislecture.

Genetic Recombination


3 different types:1) Homologous recombination• requires homologous sequences• get long regions exchanged between

homologous sequences

2) Site-specific recombination• requires a SPECIFIC short DNA sequence and a recombinase (ex. Viral genome integration)

3) Transposition (“jumping genes”)• short sequences (transposons) can excise and reinsert at a different place in the genome.

Homologous Recombination(“General recombination”)


In bacteria:

• When new DNA gets into a cell:

–by transformation: uptake of naked DNA into a bacterial cell (i.e. what happened in Griffith’s experiment when live R bacteria took up dead S bacterial DNA and were transformed)

–by conjugation: chromosome transfer (Hfr strains)

Genetic Information Can Be Transferred Between Bacteria• In 1946, Lederberg and Tatum showed

that two different strains of bacteria with different growth requirements could exchange genes

• Lederberg and Tatum surmised that the bacterial cells must interact with each other - the process is now known as sexual conjugation

Progeny cells had a combination of genetic infofrom both parents i.e. recombination had occurred

Parents must have “interacted”

Bacteria

Sexual conjugation

Fertility factor

Has Fertility (F) factor = a plasmid-small DNA circle-extrachromosomal-replicates autonomously

Transfers F plasmid to F- cell

Via a temporary bridge called a “pilus”-genes for pilus formation are on the F factor plasmid

F+(donates DNA)

F-(receives DNA)

Bacterial Conjugation

F-

F+

Pilus

1) Transfer is initiated by a “nick”Single-stranded break in F factor

2) 5’ end is transferred Through pilus to the F- cell

3) Entering F plasmid Is copied

4) conjugation converts theF- cell into an F+ cell

F factors can integrate into the host chromosome

• If an ‘F factor’ integrates it turns the host chromosome into an “Hfr chromosome” and the cell into an Hfr cell

• Hfr = “high frequency of recombination”

• Hfr cells can make pili and conjugate with F- cells

• When the F factor is transferred to F- cells adjacent genes from the chromosome are also transferred

Hfr cells can transfer host chromosomal genes

Chromosome Transfer in BacteriaFrom Fig 29.7 The F factor sequence is indicated by the triangle)

Genes are transferred in a fixed order, theoretically the whole chromosome can be transferred

Homologous Recombination(“General recombination”)


In bacteria:• When new DNA gets into a cell:

– by conjugation: chromosome transfer (Hfr strains)– by transformation: uptake of naked DNA into a bacterial cell

• During DNA repair:–Probably the most important role of recombination in bacteria– bacterial mutants with a non-functional recombination system have trouble coping with DNA damage

Homologous Recombination


In eukaryotes:

Recombination maintains genetic diversity in a population

• occurs during meiosis– when diploid germline cells divide to produce haploid gametes (ova and sperm)

• during DNA repair

DNA is the genetic material

Meiosis Diploid germ line cell

DNA replication

Exchange of genetic material

2nd cell divisionwithout DNAreplication

4 haploid gametes

“Mixing” goes on!

Recombination

Pictures of homologous recombination during meiosis

First Mechanistic Clues

• In 1961, Meselson and Weigle showed:

1) homologous recombination involves the breaking and rejoining of chromosomes (DNA replication is not required)

2) can get recombination products that are “heteroduplexes”

Meselson and Weigle• Did an experiment with differentially-labeled

bacteriophage (viruses that infect bacteria)• Used density labels instead of radioactivity

1) Prepared “heavy” phage: labeled with 13C and 15N

2) Prepared “light phage”: labeled with 12C and 14N

3) Mix both types of phage in one flask with bacteria

(under conditions which inhibit DNA replication)• injected viral DNA gets packaged into new particles

4) separate viral progeny on a density gradient

Q: Do you get intermediate density viruses?

Meselson & Weigle - part 1

Note therecombinedviral genomes

Meselson & Weigle - part 2


The process wasenhanced by UVlight treatment(causes DNAnicking)

Recovery of intermediate densityphage is proof thatrecombination occurred




Lawn of host cells

Plaque assay(1 phage infects one cell)

HeavyXYZ

Lightxyz

Intermediatedensityphage

Examine virusfrom singleplaque….

… progenyviruses sometimeshad 2 differentgenomes!

Start with two different phage genotypes: XYZ and xyz

Mechanistic clue: it’s not always just cutting and pasting (got “heteroduplex” recombinant genomes)

How you can explain the results




A single recombination experiment can give two different types of recombination

products



Splicerecombinant

Patchrecombinant

Two differentStarting genotypes

Mechanism of Recombination

• General recombination: any pair of homologous DNA segments as substrates (100% homology NOT needed)

• In 1964, Robin Holliday proposed a model involving single-stranded nicks at homologous sites

• Duplex unwinding, strand invasion and ligation create a Holliday junction

Recombination Model

1) Alignment of 2homologous DNA duplexes

2) single-strandednick occurs

3) Strand exchange/invasion

4) exchanged strandsare ligated together &form a Holliday junction

5) junction migratescausing recombinationof the two duplex DNAs

Recombination Model (continued)

6) Resolution of thejunction intermediategives either patch orsplice recombinants

Resolution of Holliday Junctions

“Patch”

“Splice”

Resolution of Holliday Junctions

To see this in 3D go to:

http://www.wisc.edu/genetics/Holliday/holliday3D.html

Requirements for Recombination

1) Initiate the process-recombination requires a single-stranded DNA

overhang

gap

Either:

-2 enzymatic activities:1) helicase: unwinds duplex DNA, ATP-dependent2) nuclease: DNA hydrolysis (breaking backbone)

-overhangs are produced by RecBCDEnzyme complex = RecB, RecC and RecD proteins

Chi sequence:5’-GCTGGTGG-3’~1000 such sites on the E. coli genome-are recombinationalhot-spots

Single-Stranded Binding protein (SSB) binds ssDNA non-specifically and protects it from degradation, and from becoming double stranded again

1) Initiate

Requirements for Recombination

2) Holliday junction formation

-by RecA

- has “recombinase” activity

- Mediates homologous base pairing(aligns 2 homologous DNA partners)

- Catalyzes strand exchange, ATP-dependent

2) Strand exchange and junction formation

The RecA Protein

• 38 kD enzyme that catalyzes ATP-dependent DNA strand exchange, leading to formation of Holliday junction

• RecA forms a helical filament with a groove to accommodate DNA

RecA protein Filament crystal structure

A single RecA = 38kD(ribbon diagram andred monomer)

Can assemble into aHelical Filament = 6 RecA’s per turn

Helical filament has agroove that can fit DNA

DNA modeled into thegroove has to have itsnormal helix extendedto 150% normal length

RecA has 2 sites forbinding to DNA:1o site and 2o site

The 1o site has ahigher affinity for DNA, so it gets filled first.

Next, the 2o site is filled by the recombination partner dsDNA. But this binding is transienti.e. the RecA is scanningalong the dsDNA.

If the single strand in the1o site can form a duplexwith one strand of the recombination partner thenthe remaining single strandgets trapped tightly in site 2.Why? A. site 2 has ahigh affinity for ssDNA

Model for DNA Structure During Strand Exchange

• Proc. Natl. Acad. Sci. USA Vol. 98, Issue 15, 8425-8432, July 17, 2001

“Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51 family of proteins: a possible advantage of DNA over RNA as genetic material”

Takehiko Shibata et al.

• used NMR to investigate structure of RecA-ssDNA complexes in solution

Mix:RecA protein ssDNA (short oligonucleotide)ATPS (non-hydrolyzable analog of ATP)

Take NMR spectrum

Concluded:• when bound to RecA the ssDNA forms a helixthat is 1.5X length of B form DNA

• when bound to RecA the ssDNA forms a helixthat is 1.5X length of B form DNA• modeled that would be same for the dsDNA• adjacent bases are too far apart to stack• so what stabilizes the helix???

Significance of the conclusions

• van der Waals interactions between the sugar2’C (methylene group) of one nucleotide and the adjacent base stabilize the helix.

• This gives rotational flexibility to the bases inthe DNA

Significance of the conclusions

• van der Waals interactions between the sugar2’C (methylene group) of one nucleotide and the adjacent base stabilize the helix.

• This gives rotational flexibility to the bases inthe DNA

RecA-bound DNA Normal B form DNA

2’ methylenegroup

RNA can’t do this because of the bulky hydroxyl group at the 2’ C

• Question: What drives the base rotation??

• Answer: Conversion of sugar puckers

Finishing Off Recombination

• RecA starts branch migration

• RuvA, RuvB, drive branch migration

• and RuvC processes the Holliday junction into recombination products

RuvA - is a specificity factor - it recognizes the junction and binds to it

RuvB - is an ATP-dependant motor- it migrates the junction

- Junction resolution is done by RuvC-an endonuclease

Efficient Branch Migration• Accomplished by a complex of RuvA/RuvB

RuvA (crystal structure was solved in 1996)

• functions as a tetramer

• binds to Holliday junction structure

• has a core of negatively charged amino acids that force apart the DNA strands at the junction center

• facilitates binding of RuvB

The RuvA tetramer

Ribbon structure Charge distribution Space-fill model + DNA

Efficient Branch Migration• Accomplished by a complex of RuvA/RuvB

RuvB = ATP-dependent helicase

• forms a ring of 6 monomers

• surrounds the heteroduplex DNA

• one RuvB ring assembles on either side of the Holliday junction

• drives migration by pulling ds DNA through the rings over the RuvA core

Migration of Holliday JunctionsTo see this in motion go to:

http://www.sdsc.edu/journals/mbb/ruva.html

The RuvC endonuclease

So much for bacteria,What about us??

• proteins with recA activity exist in eukaryotes (from yeast to mammals)

• examples are: Rad51, Rad55, Rad57, DncI

•Function in DNA repair

•Can mediate homologous strand exchange in vitro

•Form nucleoprotein filaments just like RecA

For next classWe will switch chapters

We are done with recombination(no transposons, no Immunoglobulin genes)

Please read: Chapter 30sections 30.1, 30.2, 30.3

Documents

Picture taken in 1929 of Emerson’s corn cytogenetics class