DNA Structure & Replication

• Why should I study genetics??
• Transformation

  Non-virulent bacteria can become virulent when mixed with heat killed pathogenic counterparts
• DNA (deoxyribonucleic acid)

  The active genetic principle involved in transformation
• Building blocks of DNA

  • Bases
• Nucleotides

  Joined via phosphodiester linkages, 5' - 3' direction
• Chargaff's rules

  Specific base-pairing of A-T and G-C in dsDNA through hydrogen bonds
• dsDNA "duplex"

  Detailed structure of a polynucleotide polymer
• DNA structure

  • B DNA is the main form in cells, A DNA with central hole is the main form of RNA-DNA and RNA-RNA helices
• DNA topology

  DNA can twist (helical turns) and writhe (long axis crosses over itself)
• DNA topology

  • Interwound writhe in supercoiled DNA, Toroidal writhe
• Topoisomerases

  Enzymes that can relax supercoiled DNA by introducing transient breaks, then repairing them
• Topoisomerases

  • Can decatenate, disentangle and unknot DNA
• DNA synthesis

  1. Requires deoxynucleotide triphosphates and a primer:template junction

  2. The free 3'-OH of the primer attacks the a-phosphate nucleophilically

  3. Catalysis only occurs with correct base pairing
• DNA polymerase

  • Its structure resembles a right hand, metal ions bound catalyze addition of nucleotides
• Proofreading exonuclease activity

  Fixes mispaired bases during DNA synthesis
• DNA replication

  1. Both strands replicated at the same time at the replication fork

  2. RNA primers used for initiation must be removed

  3. DNA helicases help separate the two strands

  4. Single stranded DNA-binding proteins stabilize the ssDNA

  5. Topoisomerases remove positive supercoils in front of the replication fork

  6. Sliding DNA clamps improve processivity of DNA polymerase
• Replicon model

  Requires a cis-acting replicator sequence and an initiator protein
• Replicators

  • DnaA, T antigen, ORC
• Eukaryotic chromosomes have replication origins every ~30kb
• Eukaryotic chromosomes replicate once per cell cycle, and can break if DNA replication is incomplete
• Catenanes

  Daughter DNA molecules linked together at the end of replication, decatenated by topoisomerases