chapter 13

• Biomolecular Machines


  Review and DNA Replication Preview
• Circumstantial evidence for DNA as genetic material

  • Right place: DNA stained present in nucleus
  • DNA amount is 2 times greater in somatic cells than gamete cells (eggs / sperm)
  • DNA base sequence varies between species
• DNA transformed bacteria


  Changed from nonvirulent to virulent
• Griffith's bacteria experiment


  1. Something caused bacteria to change genetically

  2. Streptococcus pneumoniae

  3. S strain kills rats, virulent, causes disease (smooth, has capsule)

  4. R strain nonvirulent (rough), does not cause disease, NO capsule
• Avery: Was it DNA that transformed bacteria from R to S?

  1. RNAse enzymes digested RNA, R transform to S

  2. Protease enzymes digested proteins, R transform to S

  3. DNAse enzymes digested DNA, R cells did NOT transform to S

  4. Positive experiment: isolated DNA from extract, DNA alone caused transformation
• Proteins


  Have 20 different amino acids and complex molecular shapes, how can DNA with only 4 nucleotides give diversity of all living organisms?
• Answer


  Sequence of nucleotides
• Hershey-Chase Experiment


  1. T2 bacteriophage = virus that infect bacteria

  2. DNA inside protein coat (capsid)

  3. Virus enters cell, hijacks cell, new viruses burst from cell

  4. Proteins labeled with radioactive Sulfur (S35)

  5. DNA labeled with radioactive phosphorus (P32)

  6. Let viruses infect bacteria, then agitated solution, remove anything outside the bacteria, don't pop open bacterial cell

  7. Bacterial cells had P32, in pellet of centrifuge

  8. Liquid supernatant had S35 proteins

  9. DNA entered infected cells, caused genetic change
• Hershey and Chase: Summary

  • Determined that our genetic material is DNA, not protein
  • Used radioactive sulfur (proteins) and phosphorus (DNA) to determine what happens to protein and DNA in T2 bacteriophage
  • Results of experiment: Phage DNA, not protein, entered host bacterial cells
  • E. coli released newly made bacteriophages that contained radiolabeled phosphorus (P32)
• Chargaff's Rule


  • Amount of adenine always equals amount of Thymine
  • Amount of guanine always equals amount of cytosine
  • Different species and different proportions of AT versus GC
• Chargaff: Summary & Practice

  • Analyzed the base composition of various organisms
  • Molecular diversity among species: DNA base composition differs between species
  • % Adenine = % Guanine, % Thymine = % Cytosine
  • Provided further evidence that DNA is our genetic material and helped explain the structure of DNA once it was determined
• A piece of DNA has 60 nucleotides


  • If 25 nucleotides are adenine, how many are thymine? How many are leftover? How many are guanine? How many are cytosine?
• DNA General Shape from X-ray Diffraction Studies

  • Pure DNA crystalizes
  • X-rays bounce off structure, so you can determine where atoms are
  • Rosalind Franklin: DNA double helix
  • Ten nucleotides per turn
  • Each turn 3.4 nm
  • 2 nm diameter for molecule
  • Sugar-phosphate bonds = outside edge of helix
• Watson & Crick Built Models Based on Information Known

  1. Per Rosalind Franklin, put sugar phosphate backbone with nucleotide bases facing interior

  2. DNA strands ANTI-PARALLEL, one facing up, other facing down

  3. Allowed 2 strands to fit together in the 2 nm diameter

  4. Each base pair had one purine & one pyrimidine

  5. Chargaff's rule

  6. Franklin's 2 nm consistent diameter for helix
• DNA Model

  • Double stranded helix
  • Sugar phosphate backbone + base pairing interior
  • Usually, right-handed helix
  • Antiparallel: one strand up, other down
  • Major and minor grooves
  • Expose outer edges of nitrogenous bases
  • Allows proteins to interact with different shapes of DNA sequences
• Chemistry of connections: DNA

  • Hydrogen bonds hold base pairs together
  • A = T has 2 hydrogen bonds
  • G ≡ C has 3 hydrogen bonds
  • Van der Waals forces between adjacent bases on same strand "Add up to be a stronger force"
  • Stack like "poker chips"
  • Covalent phosphodiester bonds link sugar to phosphate along backbone (outside) of each strand of double helix in 3'→ 5' direction
• Sugar's Carbons are Numbered: Marked at Ends of DNA Strands

  • 5' carbon has phosphate group
  • 3' end has OH group
  • 1' carbon attaches covalently to a nitrogenous base (A, T, G, or C)
  • Phosphodiester bonds attaches from 5' to 3'
  • So, 5' end of DNA has free PO4, 3' end of DNA has free OH
• DNA is Groovy!


  • Backbones are closer together on one side of double helix, this is minor groove
  • Major groove: backbones more spread out
  • Exposed edges of nucleotides chemically distinct
  • These unique structures are recognized by proteins interacting with DNA (like transcription factors - Ch7)
• Form Relates to Function: Double Helix


  • Genetic material has organism's genetic info
  • BASE SEQUENCE of DNA = genetic info
  • Variations in sequence = species differences
  • Genetic material can mutate (permanent change in linear sequence of base pairs)
  • Genetic material precisely replicated: complementary base pairing
  • Genetic material expressed as phenotype: DNA sequence determines RNA sequence determines protein amino acid sequence
• Watson, Crick, and Franklin: Summary

  • Franklin had X-ray crystallography data that indicated DNA is a double stranded helix of a specific width. She also wrote a report with her model having the bases in the interior of the DNA molecule
  • Watson and Crick put together data from various scientists including Franklin and Chargaff
  • Structure of DNA provided an explanation for its replication
• DNA can Replicate in Test Tube

  1. Deoxyribonucleoside triphosphates: dATP, dCTP, dGTP, dTTP are monomers

  2. DNA sequence = Template to match nucleotides in new strand

  3. DNA polymerase enzyme catalyzes polymerization to chain

  4. Salts + pH buffer allowed polymerization
• 3 Options for Replication of DNA

  • Semiconservative: Each parent strand is template for a new strand, each daughter strand is part old and part new along length
  • Conservative: Parent strands are templates, daughter helices are all parental or all new
  • Dispersive replication: Daughter strands are a random mix of new and parental strands throughout all daughter helices
• Meselson & Stahl's Elegant Experiment

  1. Demonstrated DNA replication was semiconservative

  2. Nitrogen 15 heavier than N 14 used in while growing bacterial cultures

  3. Parent test tube, transfer to N 14 culture and DNA replicated every 20 minutes

  4. First generation had 1 band, part N 14 and part N 15

  5. Second generation had 2 bands, so DNA Replication was semiconservative, each parent strand is a template for building new (daughter) strand
• Origin of Replication in E. coli

  Note: DNA sequences identified by lower-case, italicized letters, so Origin of Replication is ori.
• Energy from Triphosphates Power Covalent Sugar-Phosphate Bond Formation

  • Nucleotides only add FROM 5' → 3'
  • Why? OH at 3' end used to make new bond with incoming dNTP
  • 2 phosphates leave as PPi or pyrophosphate
  • Condensation is by dehydration synthesis to form the phosphodiester linkage in the sugar-phosphate backbone
• Key Players in DNA Replication

  • Helicase: Unwinds parental strands of DNA
  • Single-stranded binding protein (SSB): keeps strands separated
  • Toposisomerase: Relieves supercoiling
  • DNA polymerase III: Lays down DNA nucleotides at free 3' end using a pre-existing strand of nucleotides
  • Primase: Lays down an RNA primer
  • DNA polymerase I: Removes RNA primer and replaces it with DNA nucleotides
  • Ligase: Connects final gap in sugar phosphate backbones
• DNA Polymerase Needs Primer

  • DNA polymerase can only add to pre-existing chain (double-stranded)
  • Primase enzyme builds primer complementary to short DNA sequence
  • In most organisms, primer is RNA!
  • When primer long enough, DNA polymerase takes over
  • DNA polymerase only adds nucleotides from 5' → 3'
  • Later, RNA primer is degraded and replaced with DNA nucleotides using another DNA polymerase
  • Ligase closes the final gap in backbone
• Always Look for the Helpers

  • DNA helicase unwinds DNA template strands
  • Single-stranded binding proteins keep strands from re-forming hydrogen bonds
  • Primase builds RNA primer
  • DNA polymerase matches base pairs & covalently- binds nucleotides to new strand
• DNA Replication: Leading and Lagging Strands

  • Leading strand has new 3' end at replication fork, keeps adding bases constantly toward fork, continuous synthesis
  • Lagging strand has new 5' end at replication fork, build some 5'→ 3', bump into existing piece, let go and rejoin further down the strand, build some 5'→ 3'
• Lagging Strand Built from Okazaki Fragments


  1. Each new piece gets RNA primer, thank you, primase

  2. DNA polymerase joins, add some bases, then bumps into existing new strand

  3. In bacteria, DNA polymerase I removes RNA primer, replace with DNA nucleotides

  4. DNA ligase seals break between DNA of fragments
• Keeping It All Together: Sliding Clamp

  • DNA polymerases processive: catalyze formation of MANY phosphodiester linkages along DNA molecule
  • DNA polymerase – DNA complex stabilized by Sliding Clamp, donut shape, water spacer to DNA strand, holds in place until 50,000 nucleotides have been added to the newly formed DNA strand
• Formation of the Lagging Strand

  Created discontinuously, replication proceeds away from the replication fork, requires many primers, primer replacements, and ligations
• Replication Complex Stationary in Nucleus

  Attaches to nuclear structures, stable, DNA spools through, enters as 1 double helix, leaves as 2 double helices
• Telomere = Repeating Sequence at End of DNA Molecule

  • Human sequence TTAGGG -3', repeats 25,000 times at end of DNA
  • Keeps DNA repair mechanisms from joining ends of DNA
  • Lost at each DNA replication, when last RNA primer removed, no DNA can me made to pair with end of strand
  • Chromosomes get shorter with each division 50-200 bases, telomeres lost rather than gene sequences
• Most Cells Stop Dividing if Telomeres Too Short


  • Genes lost from end of chromosome some, cell dies
  • Stems cells have telomerase enzymes to rebuild telomeres after each division, bone marrow, gamete producing cells
  • RNA sequence template to build new DNA telomere
• Cancer Cells have Telomerase !!


  • 90% of cancers produce telomerase, stop telomerase, stop cancer cells dividing
  • Aging: shortens telomeres?
• Proofreading & Repair


  DNA polymerase proofreads base pairing and corrects errors as it replicates
• Mismatch Repair

  • Mismatch repair checks the replicated DNA again!
  • Removes any new mismatched nucleotide
  • Brings in the correct base match for old strand nucleotide
  • One type of colon cancer has damaged mismatch repair mechanism
• Excision Repair


  • Excision repair cuts out bad bases (e.g. damaged bases)
  • High energy radiation, Chemicals from environment, Spontaneous chemical changes in bases
  • Example: Thymine dimer, T=T covalent link due to UV light, messes up base pairing to new strand, so, cut out damaged base and replace with correct base
  • Can lead to Skin Cancer in Humans!