DNA Replication pt2

• Nucleic acids


  Their general role in organisms
• DNA


  Distinguishes it from RNA
• Nucleic acids


  Considered information molecules
• DNA structure


  • Double helix
  • Anti-parallel
  • Complementary base pairing
  • Base pair hydrogen bonds
  • Phosphodiester bonds

  
• Experimental techniques by scientists

  1. Fred Griffith: Transformation converts non-virulent bacterium into virulent form

  2. Avery, MacLeod & McCarty: Transforming factor pointed to DNA, not protein, as heritable material

  3. Alfred Hershey & Martha Chase: Radioactive labeling showed DNA is heritable material

  4. Erwin Chargaff: Determined DNA proportions are always A=T, G=C

  5. Rosalind Franklin, James Watson & Francis Crick: X-ray diffraction, model building determined DNA structure

  6. Matthew Meselson & Franklin Stahl: Showed how DNA replicates using density labeling, density-gradient ultracentrifugation

  
• DNA replication in prokaryotic cells

  1. Roles of single stranded binding proteins, helicase, DNA polymerase III, DNA polymerase I, primase and DNA ligase at the replication fork

  2. Semi-discontinuous

  3. Okazaki fragments

  4. Proofreading

  5. Leading and lagging strand

  
• Comparison between prokaryotic and eukaryotic DNA replication
• Cell division by mitosis with an average 3.72 x 10^13 cells in an adult (Bianconi et al. 2013)
• DNA is condensed into chromosomes
• You are coded by your DNA
• Identical twins result from the accidental split of the embryo at first stage of cell division
• General concept of DNA replication

  1. The parent molecule has two complementary strands

  2. Separation of the two DNA strands

  3. Each parental strand serves as a template for a new, complementary strand

  
• Where DNA replication starts

  1. Origin of replication (only one in prokaryotes)

  2. Replication occurs bidirectionally
• Origin of replication


  • Region rich in A-T, easier to separate
  • Protein activates initiation of DNA replication by separating the two strands

  
• Replisome


  Complex molecular machine that does DNA replication
• DNA polymerase


  Enzyme that adds nucleotides, one by one, only to the free 3' end (5' to 3' direction)
• Things needed for DNA replication

  • Nucleoside triphosphate (nucleotide)
  • Two antiparallel strands of DNA in a double helix

  
• Helicase


  Enzyme that untwists the double helix at the replication forks, separating the two parental strands
• DnaA


  Protein that first binds to the origin of replication and separates the DNA, providing binding space for helicase

  
• Single-strand binding protein (SSB)

  Binds to the unpaired bases of the DNA strands, stabilizing them until they serve as templates

  
• DNA gyrase


  Topoisomerase enzyme that cuts and untwists ahead of the replication fork to relieve strain caused by helicase
• Replisome


  Complex molecular machine that carries out DNA replication (helicase, gyrase, SSB, primase, DNA polymerase III, ligase, etc.)

  
• Primase


  RNA polymerase that synthesizes small RNA primers

  
• DNA polymerase III


  Synthesizes a complementary DNA strand from 5' to 3' direction using the template DNA strand

  
• Leading DNA strand


  Continuously synthesized
• Lagging DNA strand


  Synthesized discontinuously as Okazaki fragments
• DNA polymerase I


  Replaces the RNA nucleotides of the primers with DNA versions
• DNA ligase


  Forms phosphodiester bonds between Okazaki fragments

  
• DNA polymerase I and III have 3'-5' exonuclease activity for proofreading
• Synthesis of DNA is done simultaneously on leading and lagging strands
• The replisome contains all the enzymes necessary for DNA replication

  
• DNA polymerase epsilon


  Replicates the leading strand in eukaryotes
• DNA polymerase delta


  Replicates the lagging strand in eukaryotes
• PCNA sliding clamp


  Clamp that attaches the enzyme complex to the DNA in eukaryotes
• Eukaryotic replication is complicated by larger amount of DNA organized into multiple chromosomes and linear chromosome structure
• Origins of Replication


  Stretch of DNA with specific sequence and chromatin properties where replication initiates in eukaryotes

  
• Eukaryotic chromosomes have hundreds or thousands of replication origins, while prokaryotes have only one
• Telomeres


  Repetitive non-coding sequences at chromosome ends that protect the coding portion
• Telomere shortening is connected to cell aging, loss of telomeres may limit cell division
• Telomerase


  RNA-dependent DNA polymerase that extends the overhanging telomere strand