Biochemistry 441 Lecture 13 Ted Young March 8, 2000

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(No Transcript)
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Biochemistry 441Lecture 22Ted Young March
forth , 2009

• Topic for today
• DNA recombination
• We will only discuss homologous recombination
  (pp978-985 in your text)

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• Nothing in life is to be feared. It is only to
  be understood. Marie Curie

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Homologous genetic recombination Why recombine?

• 1. Evolutionary argument increase fitness of
  populations-experimental support from
  micro-organisms.
• 2. Molecular rationale proper segregation at
  meiosis. Experimental support mutants that cant
  recombine cant complete meiosis
• 3. DNA repair-associated recombination.
• 4. DNA replication requires recombination in some
  organsims.

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Recombination occurs during meiosis in germ-line
cells, rarely or not at all in mitotically
dividing somatic cells
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Homologous recombination starts with a
double-stranded break in the DNA

• First evidence density-labeled DNA in virus
  particles

Infect E. coli

Phage-mutant 1 aBc
Recombinant phage have inherited parental DNA
Phage-mutant 2 AbC
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Recombinant phage were hybrid in density
Phage-mutant 1 aBc-light isotopes
Phage-mutant 2 AbC-heavy isotopes

abCrecombinant phage particles
1.6
density
1.5
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Interpretation of phage cross
A b C
Parent 1-light Parent 2- heavy
density Recombinants-
X
a B c
Recombinants with inherited parental
heavy density DNA Replicated recombinants
a b C
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Homologous recombination starts with a
double-stranded break in the DNA-2

• Second line of evidence yeast integrative
  transformation is enhanced by cutting plasmid DNA
  with a restriction enzyme

Transforming DNA transformation efficiency
(tfs/mg DNA) Circular plasmid-HIS3 10 Linea
r plasmid-HIS3 105 In both cases the plasmid
had integrated at the HIS3 locus on the
chromosome.
EcoR1- cut
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Interpretation of yeast transformation experiment
Conclusion creating a double-stranded
break stimulates homologous genetic recombination
HIS3
x
His3-
x
HIS3
His3-
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Homologous recombination-at the DNA level-the
Holliday model
cut and expose
invade
extend and migrate
replace
cleave intermediate
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Homologous recombination-at the DNA level
DNA caught in the act of recombining
Bubble of single- stranded DNA as predicted by
Holliday model
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Homologous recombination-at the DNA
level-enzymology
The RecBCD enzyme complex creates a
recombinogenic 3 DNA end, starting at a
double-stranded break in the DNA.
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RecA-a universal enzyme for homologous
recombination
recA hexamer?
ss DNA
recA-DNA filament
recA monomer?
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Branch migration can also generate diversity
A G T
A G
This heteroduplex or mismatch can go on and
generate two different genotypes- There would be
a SNP at this site when the two individuals are
compared.
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RecA-promotes single-strand invasion of a duplex
DNA
RecA homologs have been found in all
organisms. RecA promotes strand exchange in
vitro and is essential for homologous recombinatio
n in vivo.
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Summary

• Homologous recombination uses enzymes with
  activities identical to those used in DNA
  replication and repair-single strand binding
  proteins, helicases, ligases, and DNA
  polymerases, as well as enzymes unique to this
  process-such as recA that promote association of
  a single strand of DNA with a duplex. Homologous
  recombination can also used to repair damaged
  chromosomes and to regenerate telomeres.