DNA Replication, Repair and Recombination

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Chapter 6

• DNA Replication, Repair and Recombination

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DNA Replication

• Must duplicate genetic information
• Must happen prior to cell division
• Must monitor the DNA to repair any damage during
  the cells life or during synthesis
• Mutations may effect the information
• permanent, accidental changes
• may benefit the organism, cause no change or be
  harmful

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DNA Template

• Each strand of parent DNA is used as a template
  to make the new daughter strand
• DNA replication makes 2 new complete double
  helices

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DNA Replication

• Semiconservative
• Daughter DNA is a double helix with 1 parent
  strand and 1 new strand
• Found that 1 strand serves as the template for
  new strand

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Replication Origin

• Site where replication begins
• 1 in E. coli
• 1,000s in human
• Strands are separated to allow replication
  machinery contact with the DNA
• Many A-T base pairs because easier to break 2
  H-bonds that 3 H-bonds
• Note anti-parallel chains

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Replication Fork

• Bidirectional movement of the DNA replication
  machinery each end of the replication fork will
  have DNA synthesis happening

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DNA Polymerase

• An enzyme
• Catalyzes the addition of nucleotide to the
  growing DNA chain as a nucleotide triphosphate
• 3OH of sugar attacks first phosphate of
  triphosphate bond on the 5 C of the new,
  incoming nucleotide
• releasing PPi energy

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DNA Polymerase

• Bidirectional synthesis of the DNA double helix
  at both forks
• Corrects mistaken base pairings proof-reading
  ability
• Requires an established polymer (small RNA
  primer) before addition of more nucleotides
• Other proteins and enzymes necessary

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Replication Fork is Asymmetrical

• Begin adding nucleotides at origin
• Add subsequent bases following pairing rules
• Expect both strands to be synthesized
  simultaneously even though run in opposite
  directions
• NOT the case

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Synthesis contd

• Actually
• Simple addition of nucleotides along one strand,
  as expected
• Start at 3 end of template
• Proceeds 5 ? 3 with respect to daughter
• Remember how the nucleotides are added!!!!!
• Called leading strand
• Other strand also 5 ? 3 daughter synthesis but
  the template is also 5 ? 3
• Many short segments later bound together
• Called lagging strand

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DNA Replication Fork Fig 6-17
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Mistakes during Replication

• Base pairing rules must be maintained
• mistake genome mutation, may have consequence
  on daughter cells
• Only correct pairings fit in the polymerase
  active site
• If wrong nucleotide is incorporated?
• polymerase uses its proofreading ability
• cleaves phosphodiester bond of improper
  nucleotide
• Activity 3 ? 5
• adds correct nucleotide and proceeds down the
  chain again in the 5 ? 3 direction

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Proofreading
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Polymerization and Exonuclease Activity

• Both activities in the polymerase protein
• polymerization in the 5 to 3 direction
• exonuclease (removal of nucleotide) in the 3 to
  5 direction

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Why 5 to 3?
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Starting Synthesis

• DNA polymerase can only ADD nucleotides to a
  growing polymer
• Another enzyme, primase, synthesizes a short RNA
  chain called a primer
• DNA/RNA hybrid
• base pairing rules followed (BUT A-U)
• later removed, replaced by DNA and the backbone
  is sealed

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Primers contd

• Simple addition of primer along leading strand
• RNA primer synthesized 5 ? 3, then can begin
  polymerization with DNA nucleotides
• Many primers are needed along lagging strand
• 1 primer per Okazaki fragment small fragments
  of new DNA made along the lagging strand

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Synthesis

• Other enzymes needed to excise (remove) the
  primers
• Nuclease breaks up the RNA primer
• Repair polymerase replace of RNA with DNA
• DNA ligase seals the sugar-phosphate backbone
  by creating phosphodiester bond

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Polymerization Needs Other Proteins

• Helicase opens double helix
• Single-strand binding proteins (SSBP) keep
  strands separated
• most abundant protein in the machine
• Sliding clamp
• Subunit of polymerase
• Helps polymerase slide along strand
• All are coordinated with one another to produce
  the growing DNA strand

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Machine at the Replication Fork
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Polymerase Proteins Coordinated

• One polymerase complex apparently synthesizes
  leading/lagging strands simultaneously
• Even more complicated in eukaryotes

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Telomeres

• Telomerase adds a repeat of nucleotide sequences
  so that the primase can add the primer to get all
  the DNA copied

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DNA Repair

• For the rare mutations occurring during
  replication
• For mutations occurring with daily assault
• If no repair
• In germ (sex) cells ? inherited diseases
• In somatic (regular) cells ? cancer

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Effect of Mutation

• The change from A to T causes a change from
  glutamic acid to valine
• Dramatic effect on RBC when oxygen levels are low

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Cancer Incidence

• Cancer increases as people age
• Gradual changes in the DNA over time
• Happens in somatic cells

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Uncorrected Replication Errors

• Mismatch repair
• enzyme complex recognizes mistake and excises
  newly-synthesized strand and fills in the correct
  pairing

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Mismatch Repair contd

• Eukaryotes label the daughter strand with a
  nick to recognize the new strand
• Occurs on leading and lagging strand by unknown
  mechanism

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Depurination or Deamination

• Depurination removal of a purine from the DNA
  strand
• Deamination is the removal of an amine group on
  Cytosine to yield Uracil
• could lead to the insertion of Adenine rather
  than Guanosine

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Chemical Modifications
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Thymine Dimers

• Caused by exposure to UV light
• 2 adjacent thymine residues become covalently
  linked

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Repair Mechanisms

• Different enzymes recognize, excise different
  mistakes
• DNA polymerase synthesizes proper strand
• DNA ligase binds new fragment with the polymer