DNA structure

• Introduction: importance of DNA strucure
  • structure of DNA proved that it was in fact the molecule of heredity
  • structure of DNA essentially proved the replication mechanism
• Chemical nature of the nucleotide
  • Composed of three components
    • Phosphoric acid
      • provides negative charge to backbone
      • participates in phosphodiester bonds
    • Pentose (furanose) sugar
      • ribose for RNA, deoxyribose for DNA
      • numbering of carbons, and important groups on 1', 2', 3', and 5' carbons
    • Nitrogenous bases
      • purines and pyrimidines:generic structures
        • numbering of atoms
        • N-glycosidic bonds
        • Adenine, guanine, cytosine, thymine, uracil
      • nomenclature
        • bases, nucleosides, nucleotides
        • adenine vs. adenosine vs. adenylate, e.g.
      • base pairs form by H-bonding between bases on opposite strands
• The double helix
  • General features
    • Pentose-P backbone winds around the outside of the cylinder
      • protects the bases buried within
      • coats helix with uniform negative charge
    • Bases are stacked inside the helix cylinder
      • numerous weak interactions hold strands together
      • positions of N-glycosidic bonds creates major and minor grooves
• Structure-function correlates
  • Denaturation and annealing
    • Denaturation of DNA is defined as the formation of single strands from double stranded DNA (as opposed to an irreversible loss of structure)
      • heat and pH extremes can cause it
      • both weaken H-bonds that normally hold together the base pairs of double helix
    • Annealing is the (much slower) reversal of denaturation
      • requires salt to neutralize replusion of single stranded sugar-P backbones
      • also requires time
    • Hypochromic effect
      • DNA or RNA absorbs UV light at 260 nm.
      • ssDNA absorbs light more readily than does dsDNA
      • therefore, you can measure denaturation as a simple increase in A at 260 nm.
    • melting curves and melting temperature
      • plots of A260 vs temp
      • Tm is the temperature at which the rise in absorbance at 260 nm is half maximal (not the same thing as "temp at which DNA is half denatured")
      • Tm and G+C content: %G+C= 2.44(Tm-69.3),where Tm is in degrees C, and assuming 0.15M NaCl and 0.15 M sodium citrate, pH 7.0
    • hybridization
      • based on annealing two types of ssDNA molecules
        • rapid vs. slow annealing, compementary strands
        • hybid strands and slow annealing
      • filter binding assays
      • heteroduplex analysis will come later
      • as will recombinant DNA
    • Superhelical forms of DNA
      • Circular and supercoiled
        • nature of supercoiling
        • enzymes
          • DNA gyrase adds negative supercoils
          • DNA topoisomerases remove negative supercoils
          • DNA replication tends to introduce positive supercoiling
        • Breathing and Z-DNA
      • Palindromes
        • reverse complement vs. palindrome
          • antiparallel, complementary base pairing means you can deduce the sequence of one strand from the other, but does NOT mean that the strands are identical in sequence
          • if the strands ARE identical, we call them palindromes: from the English term for a sentence such as "Madam I'm Adam" or "A man, a plan, a canal, Panama"
        • example:
          • GATC is a palindome
          • GACT is not
        • functions and consequences of palindromes
          • frequently found in recognition site sequences (i.e., proteins "read" the palindrome as a "word")
          • at least theoretically, can introduce struture to double helical DNA
          • can and often do influence the structure of RNA
            • terminators and antiterminators
            • RNase protection
      • Direct repeats have no structural consequence, but can serve as recognition sequences.
• Genome structures
  • Bacterial genomes
    • supercoling
    • radial loops
  • Eukaryotic genomes
    • histones
    • levels of compaction

DNA Replication

• The replication fork
  • DNA polymerases
    • 1. polymerase activity (5'->3')
    • 2. proofreading exonuclease activity (3'-5')
    • 3. nick translation activity (5'->3' exonuclease)
  • Primase
  • Helicase and SSBPs
  • Leading and lagging strands
    • leading strand animation
    • lagging strand and the logistical problem of semiconservative replication
  • Okazaki fragments
  • DNA ligase
  • Another logisitical matter-DNA pol III works as a dimer that never leaves the replication fork. The backstitch concept is not quite adequate
    • loops in lagging strand
    • animated
• Spell checkers of replication
  • editing functions of DNA polymerases
  • mismatch repair

• Replication of chromosomes, and cell division
  • theta replication
  • oriC, DnaA, and the timing of replication initiation
    • the eukaryotic cell cycle
    • the prokaryotic cell cycle
  • terC and termination
  • linear chromosomes and their consequences
    • multilple origins of replication on chromosomes
    • telomeres and telomerase