ch 11-12

• DNA Is Reproduced by Semiconservative Replication
• DNA polymerases in bacteria

  • DNA polymerase I
  • DNA polymerase II
  • DNA polymerase III
  • DNA polymerase IV
  • DNA polymerase V
• DNA polymerases


  • 5'-3' polymerization
  • Exonuclease activity 3'-5'
  • Exonuclease activity 5'-3' (only DNA polymerase I)
  • Repair/damage (DNA polymerases I, II, IV, and V)
• DNA polymerase III holoenzyme

  Composed of 10 subunits
• Key issues resolved during DNA replication

  • Unwinding of helix
  • Reduce increased coiling generated during unwinding
  • Synthesis of primer for initiation
  • Discontinuous synthesis of second strand
  • Removal of the RNA primers
  • Joining of gap-filling DNA to adjacent strand
  • Proofreading
• Unwinding of Helix


  1. DnaA binds to ORI causing conformation change

  2. DNA helicase (DnaB) assembles around exposed ssDNA

  3. Single-stranded binding proteins (SSBPs) stabilize the open conformation of helix
• Reduce Increased Coiling Generated During Unwinding

  DNA gyrase relieves DNA supercoiling
• Synthesis of Primer for Initiation


  Primase (RNA polymerase) synthesizes RNA primer
• Discontinuous Synthesis of Second Strand


  1. DNA synthesis by PolIII occurs only in the 5'->3' direction

  2. Leading strand undergoes continuous synthesis

  3. Lagging strand undergoes discontinuous synthesis as Okazaki fragments
• Primer Removal and Gap Repair

  1. DNA polymerase I removes the primers on the lagging strand

  2. DNA ligase seals nicks and joins fragments
• Proofreading and Error Correction

  DNA polymerases I, II, and III have 3' to 5' exonuclease activity that allows proofreading
• Concurrent DNA synthesis achieved on both strands at single replication fork
• Termination of DNA Replication can occur whenever two replication forks meet or at specific Ter sites
• Okasaki fragments are a consequence of the inability of the DNA polymerase to polymerize in the 3′ to 5′ direction
• Eukaryotic and bacterial DNA replication share many features
• Eukaryotic DNA polymerases


  • Pol a
  • Pol d
  • Pol e
  • Pol g
• Polymerase processivity


  Strength of the association between a polymerase and the template
• Polymerase Switching


  Pol a by Pol d or e for elongation
• Issues regarding eukaryotic cells

  • More DNA than in prokaryotic cells
  • DNA is complexed with proteins
  • Chromosomes are linear
• Eukaryotic chromosomes contain multiple ORIs
• Eukaryotic DNA complexed with binding proteins (chromatin)
• The synthesis of new histone proteins is tightly coupled to DNA synthesis during the S phase
• Replication at ends of linear chromosomes results in shortening of DNA
• Telomeres


  Long stretches of short repeating sequences
• Viral and bacterial chromosomes are relatively simple DNA molecules
• Bacterial chromosomes are compacted into a structure called a nucleoid
• Bacterial DNA is associated with several DNA binding proteins - 2 are Hu and HNS
• Hu and HNS


  Small positively charged proteins that can bond ionically to the phosphate backbone, similar to histones but not actually involved in compacting DNA
• Viral chromosomes are nucleic acid, either DNA or RNA, single or double stranded, circular or linear molecules
• Viral genetic material is inert until released into host cell
• Viral chromosomes are able to package long DNA into a small volume just like bacteria and eukaryotic cells
• Supercoiling


  Facilitates compaction of DNA
• Supercoiled DNA


  Closed-circular molecules that are more compact and sediment more rapidly than linear forms
• Linking number


  The number of times the two strands of a closed circular DNA molecule are intertwined
• Supercoils are formed in the direction opposite to the unwinding
• Topoisomers


  Two identical molecules that differ only in linking number
• Topoisomerases


  Enzymes that cut one or both DNA strands and wind or unwind the helix before resealing the ends
• During interphase, eukaryotic chromosomes uncoil and decondense into a form called chromatin
• Nucleosomes


  Repeating units of chromatin consisting of 147 bp of DNA wrapped around a histone octamer
• Histones


  Positively charged proteins associated with chromosomal DNA in eukaryotes, containing large amounts of lysine and arginine