DNA

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• DNA DNA Replication

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History

• DNA
• Comprised of genes
• In non-dividing cell nucleus as chromatin
• Protein/DNA complex
• Chromosomes form during cell division
• Duplicate to yield a full set in daughter cell

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DNA is Genetic Material
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From Chapter 2

• Nucleic acids are polymers
• Monomers are called nucleotides
• Nucleotides base sugar phosphate
• Base purine or pyrimidine
• Purines adenine, guanine
• Pyrimidines thymine, cytosine, uracil
• Sugar deoxyribose or ribose
• Phosphate, a single phosphate in DNA
• Sugar of nt 1 is linked to the phosphate of nt 2
  by a phosphodiester bond

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(No Transcript)
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Chapter 2 contd
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DNA is a Double Helix

• Nucleotides
• A, G, T, C
• Sugar and phosphate form the backbone
• Bases lie between the backbone
• Held together by H-bonds between the bases
• A-T 2 H bonds
• G-C 3 H bonds

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H - Bonds

• Base-pairing rules
• A?T only (A?U if DNA-RNA hybrid)
• G?C only
• DNA strand has directionality one end is
  different from the other end
• 2 strands are anti-parallel, run in opposite
  directions
• Complementarity results
• Important to replication

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Helical Structure
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Nucleotides as Language

• We must start to think of the nucleotides A, G,
  C and T as part of a special language the
  language of genes that we will see translated to
  the language of amino acids in proteins

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Genes as Information Transfer

• A gene is the sequence of nucleotides within a
  portion of DNA that codes for a peptide or a
  functional RNA
• Sum of all genes genome

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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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DNA Template

• Each strand of the parent DNA is used as a
  template to make the new daughter strand
• DNA replication makes 2 new complete double
  helices each with 1 old and 1 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

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DNA Polymerase

• An enzyme that catalyzes the addition of a
  nucleotide to the growing DNA chain
• Nucleotide enters as a nucleotide tri-PO4
• 3OH of sugar attacks first phosphate of tri-PO4
  bond on the 5 C of the new nucleotide
• releasing pyrophosphate (PPi) energy

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DNA Polymerase

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

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How is DNA Synthesized?

• Original theory
• Begin adding nucleotides at origin
• Add subsequent bases following pairing rules
• Expect both strands to be synthesized
  simultaneously
• This is NOT how it is accomplished

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Why DNA Isnt Synthesized 3?5
Correction Refer to Figure 6-15 on page 205 of
your textbook for corrected figure. This
figure fails to show the two terminal phosphate
groups attached on the 5 end of the nucleotide
strand located at the top of this figure.
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How is DNA Synthesized?

• Actually how DNA is synthesized
• Simple addition of nucleotides along one strand,
  as expected
• Called the leading strand
• DNA polymerase reads 3 ? 5 along the leading
  strand from the RNA primer
• Synthesis proceeds 5 ? 3 with respect to the
  new daughter strand
• Remember how the nucleotides are added!!!!! 5 ?
  3

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How is DNA Synthesized?

• Actually how DNA is synthesized
• Other daughter strand is also synthesized 5?3
  because that is only way that DNA can be
  assembled
• However the template is also being read 5?3
• Compensate for this by feeding the DNA strand
  through the polymerase, and primers and make many
  short segments that are later joined (ligated)
  together
• Called the lagging strand

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DNA Replication Fork Fig 6-12
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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 included
• Polymerase uses its proofreading ability to
  cleave the phosphodiester bond of improper
  nucleotide
• Activity 3 ? 5
• And then adds correct nucleotide and proceeds
  down the chain again in the 5 ? 3 direction

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Proofreading
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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 for this short stretch
• Base pairing rules followed (BUT A-U)
• Later removed, replaced by DNA and the backbone
  is sealed (ligated)

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Primers contd

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

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Removal of Primers

• Other enzymes needed to excise (remove) the
  primers
• Nuclease removes the RNA primer nucleotide by
  nucleotide
• Repair polymerase replaces RNA with DNA
• DNA ligase seals the sugar-phosphate backbone
  by creating phosphodiester bond
• Requires Mg2 and ATP

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Other Necessary Proteins

• Helicase opens double helix and helps it uncoil
• Single-strand binding proteins (SSBP) keep
  strands separated large amount of this protein
  required
• Sliding clamp
• Subunit of polymerase
• Helps polymerase slide along strand
• All are coordinated with one another to produce
  the growing DNA strand (protein machine)

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Components of the DNA Replication
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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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DNA Repair

• For the rare mutations occurring during
  replication that isnt caught by DNA polymerase
  proofreading
• 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
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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 nicks
  to recognize the new strand
• Separates new from old

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Depurination or Deamination

• Depurination removal of a purine base 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 on next round

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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 joins new fragment with the polymer