HCS113 DNA Structure & Replication

• Genetics


  The study of inheritance, variation & genes
• Molecular genetics is the focus here: the molecular basis of gene structure & function (expression)
• Bacterial Genetics


  • Bacteria are the majority of living organisms (approx. 4-6 x10^30 cells = 5 thousand billion metric tonnes of biomass)
  • Bacteria cause clinical problems like development of resistance to antimicrobial drugs
  • Bacteria can be industrially exploited e.g. for vitamin production, industrial enzyme production, biopharmaceuticals, bioplastics, biogas (developing world)
  • Bacteria can be used for bioremediation
  • Bacteria can undergo horizontal gene transfer which spreads antibiotic resistance and raises safety concerns for GMOs
• Gene


  A discrete genetic unit that controls a hereditary characteristic
• Allele


  Alternative forms of a gene (e.g. wild-type vs mutant) that may (or may not) change the phenotype
• DNA is the hereditary material
• DNA is like a computer hard-drive carrying the operating system & software that encodes structures & functions of the cell
• DNA


  Relatively inert (stable), but codes for every function of the cell
• DNA Structure


  • Composed of 4 types of nucleotide, each with a nitrogenous base, five-carbon sugar (deoxyribose), and phosphate (PO4) group
  • Bases: adenine, cytosine, thymine, or guanine
  • Amounts of A = T, G = C, purines = pyrimidines (Chargaff's Rule)
  • Double-stranded helix with antiparallel strands (5' to 3' direction)
  • Nucleotides linked by 3'-5' phosphodiester bonds
  • Bases on opposite strands linked by hydrogen bonding: A with T, G with C

  
• DNA replication is semiconservative: one strand acts as a template for synthesis of the other (complementary) strand
• Central Dogma


  Information flows unidirectionally from DNA (genetic template) to RNA ("expression" of gene - i.e. switched on) to protein
• In prokaryotes, transcription and translation are concurrent (happen together), while in eukaryotes they are separated (transcription in nucleus, translation in cytoplasm)
• DNA Replication


  1. DNA template

  2. Deoxynucleoside triphosphates (dNTPs)

  3. DNA polymerase complex

  4. DNA or RNA primer

  5. Divalent metal cations (Mg2+)

  
• DNA Polymerase


  • Catalyzes DNA synthesis
  • Not a single protein - a protein (enzyme) complex
  • Multiple versions (DNA polymerase I, DNA polymerase III)
  • Requires DNA/RNA primer to start
  • Adds nucleotides by complementary base pairing with template strand
  • Synthesizes DNA in 5' to 3' direction

  
• At least 160 different proteins are involved in replicating the human genome, and at least 80 genetic diseases result from mutations in these proteins or from errors in DNA replication or repair
• dGTP


  Deoxyguanosine triphosphate
• DNA polymerase


  Enzyme that catalyzes DNA replication
• Mg2+


  Magnesium ion
• DNA replication


  1. DNA replication is catalyzed by DNA polymerase enzyme

  2. DNA polymerase is not a single protein - it is a protein (enzyme) complex

  3. Multiple versions of DNA polymerase exist (DNA polymerase I, DNA polymerase III)

  4. DNA polymerase needs DNA/RNA primer to start

  5. Nucleotides are added by complementary base pairing with template strand

  6. Substrates (dNTPs) are hydrolyzed as added, releasing energy for DNA synthesis

  7. Enzymatic synthesis of DNA is ALWAYS in 5' to 3' direction - DNA polymerase adds nucleotides to the 3' end of the growing strand
• DNA polymerases catalyse formation of 3' to 5' phosphodiester bonds
• Property of all DNA polymerases

  • They can only extend pre-existing DNA (or RNA) chain by using a primer
  • They require a double-stranded template to begin

  
• For RNA polymerases to transcribe DNA


  They can start from scratch; they do not need a primer

  
• DNA replication needs a primer; extension is in 5' to 3' direction
• Transcription does not need a primer; extension is also in 5' to 3' direction
• DNA polymerases


  • Require a primer to synthesize DNA from a template
• Mechanism of DNA Replication

  1. DNA helicase unwinds the double helix

  2. Template strands are stabilized by other proteins - single-stranded DNA binding proteins make template available

  3. RNA primase catalyzes synthesis of short RNA primers, to which nucleotides are added

  4. DNA polymerase III extends strand in 5'-to-3' direction

  
• DNA helicase


  Enzyme that unwinds the double helix during DNA replication

  
• RNA primase


  Enzyme that uses single stranded DNA as template to synthesise short complementary RNA molecule (primer), providing double-stranded template for DNA pol to extend

  
• DNA replication


  1. Leading strand: copied continuously 5' to 3'

  2. Lagging strand: DNA copied in smaller stretches (Okazaki fragments)

  3. Okazaki fragments synthesised 5' to 3' direction (cannot synthesise 3' to 5')

  4. Results in lots of short sections that don't join (lack phosphodiester bond) & are interspersed with small sections of RNA

  
• DNA synthesis can never go in 3'-5' direction
• Joining Okazaki Fragments


  1. On lagging strand, DNA pol III can only synthesise DNA until RNA-primed start of next Okazaki fragment

  2. DNA pol III has no 5'-3' exonuclease activity so stops & falls off DNA

  3. DNA pol I takes over & removes RNA using 5'-3' exonuclease ability, replaces RNA with DNA

  4. Leaves gap between fragments, without phosphodiester bond

  5. Phosphodiester bond completed by enzyme DNA ligase
• DNA replication is a multi-protein process
• There is a gap in the phosphodiester backbone during lagging strand synthesis
• RNA primase provides the primer for DNA polymerase III to extend
• DNA replication is discontinuous on the lagging strand