DNA Replication and Repair

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DNA Replication and Repair

• A Structural Biological Viewpoint

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The Point of Structural Biology

• Structural biology exists to provide a framework
  to organize the results of biochemical
  experiments.
• Structural biology does not generally prove a
  mechanism of action but provides likely
  possibilities which need to be pursued.

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SB of DNA Replication

• PCNA is the central actor at the replication
  fork. We will look at 2 different aspects.
• The interaction of PCNA with DNA and proteins.
• Loading of PCNA onto DNA.

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Reading

• Bambara et al., J. Biol.Chem., 1997, 272, 8,
  p.4647
• Chapados et al., 2004, Cell, 116, p. 39
• Krishna et al., 1994, Cell, 79,7, p.1233
• Bowman et al., Nature, 2004, 429, p.724 - 730

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The Replication Fork
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Enzymes of the Replication Fork
Leading Strand
Lagging Strand
Removal of the initiator RNA.
Completion of the lagging strand.
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Proteins that bind PCNA
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Damaged DNA blocks the replication fork
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Types of DNA damage
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Amount of Damage

• 100-500 cytosine deamination/cell/day
• 600 adenine methylations
• 2,000-10,000 purine losses

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Types of ssDNA Repair

• BER DNA glycosylase endogenous damage base
  modification or loss, remove base AP site. AP
  endo cuts out deoxyribose and pol fills in. Large
  of glycosylases that are highly specific for
  certain types of lesions.
• NER exogenous damage UV, bulky adducts from
  chemicals. Limited number of proteins, broad
  substrate specificity.
• MMR mismatches or small loops created during
  replication.
• RR use of the sister chromatid
• TLS damage bypass during DNA replication.

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Mismatch Repair
Template and daughter strands are discriminated
by methylation.
Prokaryotes - Eukaryotes MutS -gt MSH2,3,6 MutL -gt
MLH1,PMS1
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Nucleotide Excision Repair
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The Relationship between the Cell-Cycle and
different forms of DNA Repair
Growth 1 2n Excision Repair NHEJ
Synthesis 2n ? 4n TLS Rec. repair
Mitosis 4n ? 2n Rec. repair
The Cell Cycle
Growth 2 4n Mismatch repair Excision
repair Recombination repair
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Efficient Synthesis of DNA or How Lupus helped
unravel the mystery of the replication fork.
dA500dT10 201
Prelich et al., Nature, 1987, 326, p. 1987
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What else was known about PCNA?

• bound tightly to circular dsDNA but immediately
  fell of when linearized
• required a 5 protein complex called RFC, and ATP
  to bind DNA
• What is the explanation for these unusual
  observations?

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PCNA is a sliding clamp!
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PCNA Surface and e- potential
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The subunit interface is composed of an
intermolecular b-sheet.
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A remarkable structural similarity between
eukaryotic and prokaryotic clamps!
Bacterial homodimer
Eukaryotic homotrimer
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So, how do all these proteins bind to PCNA?
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-The RFC Complex- A member of the AAA family.
- AAAATPases couple ATP hydrolysis to
protein-protein interactions. - Most AAAATPases
are hexamers. Each subunit generally pokes an
arginine finger into the ATPase site of a
neighbouring subunit.
Well, how does the closed PCNA get onto DNA then?
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RFC Recognizes the Primer Template Junction
Tsurimoto and Stillman, JBC, 1991, 266,p.1950
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A schematic view of clamp loaders
RFC
Collar
AAA domains
PCNA
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The RFC, PCNA, DNA Complex
Collar
AAA ATPase
PCNA
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The structure of the RFC-PCNA complex
Helical, hydrophobic interaction with PCNA.
PCNA is closed.
RFC forms a right handed spiral!
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And the multimedia version!
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The RFC spiral is tilted w/r to PCNA.
The AAA module of RFC-A is related to that of
RFC-B by a 61o rotation and 5.5Å translation.
This closely matches the 5.6Å rise per 60o
rotation of double-helical B-form DNA.
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The Interaction with ATP
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A model for the interaction of RFC and DNA
N-terminus of a helix
Interaction primarily with minor groove. Only
single stranded DNA fits in collar.
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Details of the RFC-DNA interaction
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The Final Pieces
The nucleotide free, inactive form.
Model with the xtal structure of a real
primer-template junction. The junction of A and B
DNA causes a 40o bend in the helix which allows
it to fit the angle between RFC and PCNA.
A DNA
B DNA
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EM-A crystallography alternative
Miyata et al., NSB, 2004, 11,7, p.632
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RFC-PCNA Still to Come

• How is the ring opened?
• In bacteria this is partially understood.

Jerulzami et al, Cell, 2001, 106, p.417 following
figures as well
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The interaction with the clamp loosens the
subunit interface.The interface of the closed
clamp causes a distortion in an a-helix. This
acts like a spring that is released by the
binding of the clamp loader. The clamp loader
also undergoes dramatic conformational changes
upon binding to the clamp, forcing a hydrophobic
wedge (yellow) up against the clamp.
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Details of the clamp-clamp loader interaction
loader
clamp
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One Subunit of the clamp in the clamp-clamp
loader complex is straighter.
ssDNA, but not dsDNA can pass through.
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Conformational Changes in the Clamp Loader
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Opening of the Clamp
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Recent Work in Eukaryotes Confirms and Extends
these Findings 1. Experimental Data
2D micrographs of individual RFC-PCNA complexes
bound to DNA.
3D reconstructions using multiple 2D images and
tomography.
Electron micrographs of RFC-PCNA Complex from
Archaebacteria. Resolution 12Å. Miyata et al,
PNAS, 2005, 102, p.13795 (following figure as
well)
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Fitting of Subunits of Xtal structure in EM
Density
RFC
PCNA
Longitudinal slice
RFC
DNA
Arrow indicates in-plane and twisting motions in
PCNA opening.
PCNA
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Recent Work in Eukaryotes Confirms and Extends
these Findings 1. Computational Findings
This graph shows that the individual domains of
PCNA dont change conformation but that they move
with respect to each other.
This graph shows that the individual domains of
PCNA move with respect to each other, but that
the inter-domain movements are even
greater. Superposition of 1 subunit at point
indicated in simulation. Superposition of
1subunit with the second subunit at point
indicated in simulation.
Kazmirski et al., 2005, PNAS, v.102,
p.13801 Following slide as well.
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What do the simulated structures look like?
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Take-Home Lessons

• Theres more than one way to skin a cat. The same
  3D structure and hence function of a protein, can
  be achieved with completely unrelated amino acid
  sequences. Example PCNA, the b subunit of
  bacterial pol III.
• Biochemical data and structural data should be
  mutually supportive.
• Cells have evolved complex machines to
  efficiently and accurately replicate their
  genomes.
• The circular, sliding clampis a central
  component of the replication machinery in all
  life forms. It is not only the binding site for
  many enzymes involved in DNA metabolism, but also
  the site of regulation integrating DNA
  replication with the cell cycle.
• The structure of the sliding clamp is exquisitely
  tuned to allow it to be loaded onto DNA and then
  to stay bound to DNA for very long periods. It
  also allows for non-specific interactions with
  DNA but a variety of highly specific interactions
  with proteins.

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NHEJ vs. HR

• HR
• Uses homologous sequences as a template to
  resynthesize DNA
• Losseless process (in terms of the DNA sequences
  adjacent to break)
• Can result in loss of heterozygosity therefore
  mostly carried out between sister chromatids

• Non-Homologous end joining
• Joins any two ends available
• Small loss of sequence information
• Can cause loss of context inf.
• Used intentionally for generation of antibody
  diversity (VDJ recom.)

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NER in Eukaryotes