ch 14 dna

• Griffith's experiment


  1. Infected mice with S and R strains

  2. Observed that heat-killed S strain + live R strain killed mice

  
• Transformation


  Information specifying virulence passed from dead S strain cells into live R strain cells

  
• Genetic material was actually transferred between the cells
• Avery, MacLeod, & McCarty experiment

  1. Repeated Griffith's experiment using purified cell extracts

  2. Removal of all protein did not destroy transforming ability

  3. DNA-digesting enzymes destroyed all transforming ability
• Supported DNA as the genetic material
• Hershey & Chase experiment

  1. Bacteriophage DNA was labeled with 32P

  2. Bacteriophage protein was labeled with 35S

  3. Only the bacteriophage DNA (as indicated by the 32P) entered the bacteria and was used to produce more bacteriophage

  
• Conclusion: DNA is the genetic material
• DNA


  A nucleic acid composed of nucleotides with a 5-carbon sugar called deoxyribose, a phosphate group, and a nitrogenous base (adenine, thymine, cytosine, guanine)

  
• Phosphodiester bond


  Bond between adjacent nucleotides, formed between the phosphate group of one nucleotide and the 3' -OH of the next nucleotide

  
• Chargaff's Rules


  • Amount of adenine = amount of thymine
  • Amount of cytosine = amount of guanine
  • Ratio of G-C to A-T varies with different species

  
• Rosalind Franklin discovered that DNA is helical using X-ray diffraction studies
• Watson and Crick deduced the structure of DNA using evidence from Chargaff, Franklin, and others

  
• Double helix


  Two strands of DNA arranged in a double helix, with the strands running antiparallel and held together by hydrogen bonds between complementary base pairs (A-T, G-C)

  
• Three possible models of DNA replication

  • Conservative model
  • Semiconservative model
  • Dispersive model

  
• Meselson and Stahl experiment

  1. Bacterial cells were grown in heavy 15N isotope

  2. Cells were switched to media containing lighter 14N

  3. DNA was extracted and observed to support the semiconservative model of DNA replication

  
• DNA replication


  Requires a parental DNA molecule, enzymes, and nucleoside triphosphates as building blocks

  
• Stages of DNA replication

  1. Initiation

  2. Elongation

  3. Termination

  
• DNA polymerase


  • Adds new bases to the 3' end of existing strands, synthesizes in 5'-to-3' direction, requires a primer of RNA to have a free 3' end

  
• Action of DNA polymerase

  Adds complementary nucleotides to the template strand, cleaving off two phosphates from the nucleoside triphosphate

  
• Prokaryotic DNA replication (E. coli model)


  • Single circular molecule of DNA
  • Replication begins at the origin and proceeds bidirectionally around the chromosome

  
• E. coli DNA polymerases

  • DNA polymerase I
  • DNA polymerase II
  • DNA polymerase III
• Enzymes involved in DNA replication

  • Helicases unwind DNA
  • Single-strand-binding proteins coat and protect strands
  • Topoisomerases prevent supercoiling

  
• Replication is semi-discontinuous


  Leading strand synthesized continuously, lagging strand synthesized discontinuously in Okazaki fragments

  
• Synthesis at the replication fork

  1. Partial opening of helix forms replication fork

  2. DNA primase makes RNA primer

  
• Leading-strand synthesis


  • Single priming event, extended by DNA Pol III with processivity aided by the β subunit "sliding clamp"

  
• Lagging-strand synthesis


  Discontinuous synthesis, requires primase to make RNA primers, DNA Pol III to synthesize Okazaki fragments, DNA Pol I to remove RNA primers and replace with DNA, and DNA ligase to seal the backbone

  
• Synthesis at the replication fork


  1. Partial opening of helix forms replication fork

  2. DNA primase - RNA polymerase that makes RNA primer

  3. Later RNA will be removed and replaced with DNA (by Pol I)

  
• Leading-strand synthesis


  1. Single priming event

  2. Strand extended by DNA Pol III

  3. Processivity - ability of the pol to remain attached to the template

  4. β subunit forms "sliding clamp" to keep DNA Pol III attached to DNA

  
• Lagging-strand synthesis


  1. Discontinuous synthesis

  2. Primase - Makes RNA primer for each Okazaki fragment

  3. DNA Pol III (like leading strand) synthesize the Okazaki fragments

  4. DNA Pol I - Removes all RNA primers and replaces with DNA

  5. DNA ligase - Seals backbone

  
• Termination


  1. Occurs at specific site located roughly opposite the origin of replication on the circular chromosome

  2. The last stages of replication produce two daughter molecules that are intertwined like two rings in a chain. These intertwined molecules are unlinked by DNA gyrase

  
• Eukaryotic Replication


  • Larger amount of DNA in multiple chromosomes
  • Complex packaging
  • Linear structure

  
• Eukaryotic replication


  1. Requires new enzymatic activity for dealing with ends

  2. Multiple replicons - multiple origins of replications for each chromosome

  3. Not sequence specific; can be adjusted

  4. Example: early in development when cells divide rapidly, more origins can be used

  
• The eukaryotic replication fork

  1. Before S phase, helicases are loaded onto possible replication origins, but not activated

  2. During S phase, a subset of these are activated, and the rest of the replisome assembled

  3. Priming uses a complex of both DNA polymerase α and primase

  4. DNA polymerase epsilon (Pol ε) synthesizes leading strand

  5. DNA polymerase delta (Pol δ) synthesizes lagging strand

  
• Archaeal and eukaryotic replication proteins


  • Enzymes that are similar between eukaryotes and archaea, but different from those in bacteria: DNA polymerases, Replicative helicases, Primases
• Telomeres


  • Specialized structures found on the ends of eukaryotic chromosomes
  • Composed of specific sequences
  • Protect ends of chromosomes from nucleases
  • Maintain the integrity of linear chromosomes
• Replication of the end of linear DNA

  1. The last primer removed from the 3′ end of the lagging strand cannot be replaced

  2. Telomerase is an enzyme that synthesizes the telomere repeat sequences

  3. Uses an internal RNA template (not the DNA itself)

  
• Telomerase activity


  • High in early development/childhood
  • Low in most somatic adult cells
  • Exceptions: cells that keep dividing
  • Plays a role in senescence/aging
  • Contributes to cancer as cancer cells generally show activation of telomerase to maintain telomere length
• DNA damage


  • Errors due to replication
  • Mutagens - any agent that increases the number of mutations above background level
  • Importance of DNA repair is indicated by the multiplicity of repair systems that have been discovered

  
• Categories of DNA repair

  • Specific repair - Targets a single kind of lesion in DNA and repairs only that damage
  • Nonspecific - Use a single mechanism to repair multiple kinds of lesions in DNA
• Mismatch repair


  1. Removes incorrect bases incorporated during DNA replication

  2. Replaces them with the correct base by copying the template strand

  3. Must distinguish between the template strand and the newly synthesized strand