FCH 532 Lecture 14

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FCH 532 Lecture 14

• Quiz Friday on nucleic acid structures, base
  pairing
• Extra credit assignment for Friday March
  2-Seminar Speaker Matt DeLisa
• Chapter 30

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SOS response

• SOS response causes cells to stop dividing and
  repair damaged DNA.
• LexA and RecA mutants always have the SOS
  response on.
• When E. coli is exposed to agents that damage
  DNA, RecA mediates proteolytic cleavage of LexA.
  This is induced by RecA binding to ssDNA.
• LexA is a repressor of 43 genes involved in DNA
  repair (all proceeded by 20 nt sequence called
  the SOS box).

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Figure 30-59 Regulation of the SOS response in E.
coli.
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SOS Repair

• E. coli Pol III is unable to replicate through
  lesions (AP sites, thymine dimers), causing a
  replication fork collapse
• To restore the replication fork, can either
  induce recombination repair which uses a
  homologous chromosome as the template or SOS
  repair.
• Uses 2 bypass DNA polymreases (Pol IV and PolV).
• These are error-prone DNA polymerases (lack the
  3 ? 5 exonuclease)
• SOS is a mutagenic process. This is a last
  resort if DNA has not been repaired by other
  mechanisms.

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Double-strand break (DSB) repair

• Double-strand breaks (DSBs) in DNA are produced
  by ionizing radiation and free radical products
  of oxidative metabolism.
• Can also occur as intermediates in meiosis.
• Unrepaired DSBs can lead to cancer or cell death.
• 2 modes to repair DSBs
• 1. Recombination repair
• 2. Nonhomologous end-joining (NHEJ)

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Nonhomologous end-joining (NHEJ)

• Broken ends are aligned and frayed ends are
  trimmed or filled in, and their strands ligated.
• NHEJ in eukaryotes has several proteins
• Ku-heterodimer of Ku70 and Ku80
• DNA ligase IV
• Xrcc4
• Ku binds to double-stranded breaks binding to
  DNAs major and minor grooves.
• Nucleotide trimming is carried out by
  ATP-dependent Mre11 complex.
• Gaps filled in by DNA polymerases and sealed by
  DNA ligase IV and Xrcc4.

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Figure 30-62 Schematic diagram of nonhomologous
end-joining (NHEJ).
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Carcinogens

• Carcinogens-chemical agents that can cause cancer
• Up to 80 caused by exposure to these agents
• Usually damage DNA likely to induce SOS
  response, so they are indirect mutagenic agents.
• High correlation between mutagenesis and
  carcinogenesis.
• Ames Test-assays for carcinogenicity
• Salmonella typhimurium his- strain- must be grown
  in presence of His.
• Lack lipopolysaccaride coats and are highly
  permeable to many substances.
• Inactivated excision repair systems.
• Look for reversion to his phenotype.

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Figure 30-63 The Ames test for mutagenesis.
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Ames Test

• 109 cells spread on plate lacking His.
• Use a mixture of his- strains to detect base
  changes, insertions and deletions.
• Mutagen is place in culture medium that causes
  some his- strains to revert to his
• Mutagenicity is scored as of colonies less the
  few spontaneously revertant colonies that occur
  in the absence of the mutagen.
• Many noncarcinogens are converted to carcinogens
  in liver.
• Small amount of rat liver is added to Ames test
  to mimic mammalian metaboilsm.

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Homologous recombination

• Defined as the exchange of homologous segments
  between two DNA molecules.
• Bacteria are haploid and acquire foreign DNA
  through conjugation in which DNA is directly
  transferred from one cell to another via a
  cytoplasmic bridge.
• Holliday junction - corresponding strands of two
  aligned homologous DNA duplexes are nicked and
  the nicked strands cross over to pair with the
  nearly complementary strands of the homologous
  duplex after which the nicks are sealed.

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Figure 30-64 The Holliday model of homologous
recombination between homologous DNA duplexes.
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Formation of Holliday Junction
Branch migration-4 strands exchange base pairing
partners
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Resolution of Holliday Junction occurs in 2 ways

1. The cleavage of the strands that did not cross
   over exchanges the ends of the original duplexes
   to form, after nick sealing, traditional
   recombinant DNA

2. The cleavage of strands that crossed over
exchanges a pair of homologous single-stranded
segments.
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Figure 30-65a Secondary structure of the
four-stranded Holliday junction of
self-complementary decameric DNA d(CCGGTACCGG).
Watson-Crick base pairing interactions are shown
in black. 2-fold axis relating two helices of
stacked-X conformation is represented by large
black lenticular symbol.
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X-ray structure of the cross over event.
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Figure 30-66 Homologous recombination between two
circular DNA duplexes. This process can result
either in two circles of the original sizes or in
a single composite circle.
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Figure 30-67a Electron micrographs of
intermediates in the homologous recombination of
two plasmids. (a) A figure-8 structure. This
corresponds to Fig. 30-66d.
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Figure 30-67b Electron micrographs of
intermediates in the homologous recombination of
two plasmids. (b) A chi structure that results
from the treatment of a figure-8 structure with a
restriction endonuclease.
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Homologous recombination catalyzed by RecA in E.
coli

• recA- E.coli have 104-fold lower recombination
  rate than wild-type cells.
• RecA polymerizes cooperatively on ssDNA or dsDNA
  that has a single-stranded gap.
• Resulting filaments may contain 1000s of RecA
  molecules.
• Bind homologous dsDNA and using ATP catalyze
  strand exchange.

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Figure 30-68 An electron microscopybased image
(transparent surface) of an E. coli
RecAdsDNAATP filament.
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Figure 30-69a X-Ray structure of E. coli RecA
protein. Monomers yellow and blue, ADPs red. (a)
View normal to the helix axis as in Fig. 30-68.
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Figure 30-69b X-Ray structure of E. coli RecA
protein. (b) View nearly parallel to the helix
axis showing one turn of the helix.
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Figure 30-70 A model for RecA-mediated pairing
and strand exchange between a single-stranded and
a duplex DNA.

1. ssDNA binds to RecA to form initiation complex.
2. dsDNA binds to the initiation complex to form a
   transient 3-stranded helix to mediate correct
   pairing to homologous strand.
3. RecA rotates the bases of the aligned
   homologousstrands to effect strand exchange in an
   ATP-driven process.

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Figure 30-71 The RecA-catalyzed assimilation of a
single-stranded circle by a dsDNA can occur only
if the dsDNA has a 3 end that can base pair with
the circle (red strand).
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Figure 30-72 A hypothetical model for the
RecA-mediated strand exchange reaction.
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Recombination repair

• Damaged replication forks occur at a frequency of
  at least once per bacterial cell generation and
  10 X per eukaryotic cell cycle.
• DNA lesions that damage replication forks can be
  repaired through recombination repair.
• Happens when a replication fork encounters an
  unrepaired single-strand lesion.

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Figure 30-77 The recombination repair for a
single-strand lesion.

1. DNA replication is stopped at the lesion but
   continues on the opposing undamaged strand before
   replisome collapses.
2. Replication fork changes to a Holliday junction
   (Chicken Foot).
3. Single-strand gap at collapsed replication fork
   now an overhang is filled in by Pol I
4. Reverse branch migration mediated by RuvAB or
   RecG yields a reconstituted replication fork.

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Figure 30-78 The recombination repair for a
single-strand nick.

1. Single stranded nick causes replication fork to
   collapse.
2. Repair process RecBCD and RecA invasion of newly
   synthesized and undamaged 3-ending strand into
   homologous dsDNA
3. Branch migration via RuvAB makes Holliday
   junction to exchange 3-ending strands.
4. RuvC resolves Hollidayh junction making the 5
   end strand nick becomes a 5 endo fo Okazaki
   fragment.

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Figure 30-79 The repair DSB in DNA by homologous
end-joining.

1. DSB double-stranded ends are changed to
   single-stranded ends. The 3-ending strands
   pairs invades the homologous chromosme to form a
   pair of Holliday junctions.
2. DNA synthesis and ligation to fill gaps and seal
3. Both Holliday junctions are resolved.

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