Evidence for DNA as the genetic material

• Early in this century, little known about DNA; regarded as uninteresting junk
• Proteins were thought to be the only truly complex molecules in cells, and therefore must be responsible for heredity
• 1928: Frederick Griffith discovered phenomenon of transformation in bacteria
  1. Used organism Streptococcus pneumoniae.
  2. Strep bacillus has two forms:
     1. slimy colonies (S strain) forms mucous capsules, survives attack by macrophages in lung, kills mice
     2. rough colonies (R strain) lacks capsules, quickly killed by macrophage, no disease
  3. When Griffith mixed heat-killed S-strain with live R-strain, resulting organisms killed mice, and lungs were filled with S-strain.
  4. View diagram of Griffith experiment
  5. Conclusion: some chemical is surviving heat treatment, retains genetic information, is able to transmit that information to some R-strain bacteria, convert them to S. Griffith didn't know what was responsible.
• 1944: Avery, McCarty and MacLeod demonstrate that DNA is responsible for transformation in bacteria
  1. Fractionated different chemicals in S-strain bacteria, tested each separately to see what would cause transformation.
  2. Isolated DNA could transform, but no other isolated fraction could (RNA, protein, lipids, polysaccharides). Conclusion: DNA is transforming principle
  3. But critics attacked slight (less than 1%) presence of protein in the DNA extracts used, claimed this might be responsible for transformation
• 1952: Hershey & Chase prove that only DNA is responsible for bacterial virus infection of host cells.
  1. Viruses (called phage if host cells are bacteria) are much simpler than cells, contain only DNA & protein.
  2. Hershey & Chase were able to use different radioactive isotopes to distinguish DNA from protein: for DNA, used P-32 (lots of P in DNA, but none in protein); for protein, used S-35 (proteins contain S in certain amino acids, but DNA lacks S).
  3. H&C grew phage in hosts with either P or S radioisotope. Then infected different bacteria for short time, vortexed in blendor to separate phage coats from cells, and separated phage (very small) from cells (larger) by centrifugation.
  4. Result: only P-32 isotope found in cells. All S-35 could be knocked loose by blending, but cells were still infected and produced new phage. Therefore only DNA, not protein, was responsible for inheritance.
  5. View diagrams of H&C experiment

Structure of DNA

• DNA known to contain purine & pyrimidine bases, deoxyribose, and phosphate -- but how are they arranged?
• 1947: Chargaff published data showing that % of A, T, C, and G showed certain regularities
  1. % of bases varies from organism to organism
  2. % A = % T, and % C = % G. This is called Chargaff's rule. What did it mean?
• 1952: X-ray pictures of DNA taken by Rosalind Franklin in Wilkins lab in London showed some kind of helix.
• 1953: Watson & Crick published a model built from Franklin data = double helix.
  
  
  
  Suggested that Chargaff's rule was due to base-pairing of A with T, C with G.
• Interact with a DNA molecule: DNA tutorial (requires CHIME plug-in)
• In linear molecule, one strand has free 3'-end, where the other (complementary) strand has 5'-end.
• View 3' and 5' ends of DNA (protected)
• Two chains of DNA face in opposite directions, called antiparallel (protected) (defined by which way 3' and 5' sides of sugar molecule are facing). In linear molecule, one strand has free 3'-end, where the other (complementary) strand has 5'-end.
  

  5'-CAGCTAGAGTCATCG-3'
  3'-GTCGATCTCAGTAGC-5'
  

• W&C also suggested simple model for replication: if double stranded DNA uncoiled, each strand could serve as template for replication of new DNA. This was an exciting experimental prediction, and many labs set out to try to prove it.

Replication of DNA

• First enzyme isolated by Kornberg ( Nobel prize): DNA polymerase.
  1. Reaction: [dATP, dCTP, dGTP, dTTP] new DNA + P~P (pyrophosphate)
     reaction requires DNA polymerase, Mg⁺⁺, template DNA
  2. Note 1: P_i ~ P_i is immediately split into 2 P_i (inorganic phosphate ions).
  3. Note 2: energy for forming new sugar-phosphate bond comes from splitting a high-energy phosphate bond as P_i ~ P_i is removed. This always occurs at free 3'-OH group on deoxyribose (and on ribose in RNA synthesis). All nucleic acids grown by addition at 3'-end, not at 5'-end. Often referred to as 5' 3' synthesis.
  4. View animation of DNA synthesis (protected)
• Eventually discovered that cells have a variety of DNA polymerase enzymes; some serve for DNA repair rather than for new synthesis.
• Other enzymes and proteins involved:
  1. DNA helicase: unwinds DNA in front of opening replication fork (otherwise DNA would quickly tangle). Uses ATP, makes single-stranded cut, allows one strand to swivel freely around the other.
  2. Single-stranded DNA binding proteins: bind to separated DNA strands, prevent from base-pairing back together
  3. View QT movie showing unwinding of template DNA and stabilization by binding proteins (Campbell website activity)
  4. RNA primase: DNA polymerase III cannot start a growing chain from scratch; needs a short primer (a few nucleotides) to add to. This is carried out by DNA-dependent RNA primase, makes very short piece of RNA by base-pairing RNA nucleotides with template DNA.
  5. View QT movie showing synthesis of RNA primer (Campbell website activity)
  6. DNA polymerase : adds new nucleotides at free 3'ends of growing chain, uses base-pairing rules to insert complementary nucleotides (A opposite T, G opposite C, etc.) Can keep on adding indefinitely for millions of nucleotides if not blockage. Also removes RNA primers, fills in gaps by base pairing, inserts new DNA nucleotides to replace RNA primer. (several types of this enzyme)
  7. View QT movie showing DNA elongation by DNA polymerase (Campbell website activity)
  8. DNA ligase: seals any gaps where adjacent nucleotides on one strand have not been covalently joined.
• Note: many gaps result on lagging strand (see below), so lots of need for enzymes (5) and (6).

Leading and Lagging strands

• Since two strands in DNA are antiparallel, new DNA must be synthesized in opposite directions on the two template strands.
• But overall, DNA must unwind in one direction (at replication fork), overall DNA synthesis has one direction.
• No problem for the strand growing in same direction as unwinding = leading strand. Can make one long, continuous piece of DNA
• Big problem for strand growing in opposite direction to unwinding = lagging strand; must grow away from unwinding. As new template is opened up by DNA unwinding, will have to start a new copy.
• In fact, just this situation was discovered experimentally by Okazaki; found many short DNA fragments newly synthesized from lagging strand = Okazaki fragments. Must be joined together by DNA ligase to make continuous DNA strand.
• View QT movie showing overview of lagging strand synthesis (Campbell website activity)
• View QT movie showing enzymes involved in lagging strand synthesis (Campbell website activity)
• View QT movie showing both leading and lagging strand synthesis proceeding simultaneously (Campbell website activity)

DNA repair

• Any damage to DNA would be lethal. Cells often spend much more energy repairing DNA than synthesizing it.
  1. Correcting damage due to enviromental effects
     • Example: UV light thymine dimers. Energy in UV links thymine where it occurs side-by-side on one strand of DNA, screws up the ability of this bit of DNA to serve as template for replication or for correct reading of proteins.
     • One good 4-hour day at beach 10 UV-induced errors in DNA of every skin cell
     • Your skin cells spend lots of energy patrolling DNA, detecting such errors, cutting them out, and using the remaining good strand as a template for repair synthesis.
  2. Correcting errors during replication (proofreading)
     • When new DNA is synthesized, occasional errors in base pairing occur with frequency ~ 1 in 10,000 base pairs
     • If not corrected, could lead to mutations, loss of functions, loss of competitiveness, evolutionary weeding out.
     • Proofreading carried out by DNA polymerases enzymes; if base mismatch spotted, cut out new bases (keep track of which is template strand and which is new strand during replication), resynthesize copy strand from that neighborhood of template.