chapter 6

• DNA Replication


  The fundamental process where a cell copies its genetic information carried in DNA before cell division to produce two genetically identical daughter cells
• DNA Repair


  The process where a cell continuously monitors and repairs damage to its genetic material (DNA)
• For a cell to survive and proliferate in a chaotic environment, it must be able to accurately copy the vast quantity of genetic information carried in its DNA
• DNA replication must occur before a cell can divide to produce two genetically identical daughter cells
• A cell must continuously monitor and repair its genetic material (DNA), as DNA is subjected to unavoidable damage by chemicals, radiation, and reactive molecules
• Despite molecular safeguards, permanent changes (mutations) sometimes do occur in DNA
• Most mutations do not affect the organism in any noticeable way, but some have profound consequences
• Mutations can make bacteria resistant to antibiotics, and changes in DNA sequence can produce small variations that underlie differences between individuals of the same species
• Unfortunately, as mutations occur randomly, they are more likely to be detrimental than beneficial, and are responsible for thousands of human diseases, including cancer
• The survival of a cell or organism depends on keeping the changes in its DNA to a minimum
• Without the systems that are continually inspecting and repairing damage to DNA, it is questionable whether life could exist at all
• Base-pairing


  The ability of each strand of a DNA molecule to act as a template for producing a complementary strand
• DNA replication produces two complete double helices from the original DNA molecule, with each new DNA helix being identical in nucleotide sequence (except for rare copying errors) to the original DNA double helix
• Semiconservative replication


  Each daughter DNA double helix ends up with one of the original (old) strands plus one strand that is completely new
• DNA synthesis begins at replication origins, where initiator proteins bind and pry the two DNA strands apart to expose the nucleotide bases
• Replication origins are DNA sequences that are especially easy to open due to being rich in A-T base pairs
• A bacterial genome typically has a single replication origin, while the human genome has approximately 10,000 replication origins
• At each replication origin, two replication forks are formed, with DNA synthesis occurring in both directions away from the origin
• Isolating 15N-DNA and 14N-DNA

  1. Isolate 15N-DNA and load into centrifuge tube

  2. Isolate 14N-DNA and load into centrifuge tube
• Centrifugation in cesium chloride gradient

  1. Centrifuge at high speed for 48h to form cesium chloride density gradient

  2. Heavy 15N-DNA forms a high-density band, closer to the bottom of the tube

  3. Light 14N-DNA forms a low-density band, closer to the top of the tube
• Bacteria grown in 15N-containing medium


  Heavy DNA molecules
• Bacteria grown in 14N-containing medium

  Light DNA molecules
• Transferring bacteria from heavy to light medium

  Bacteria grown an additional 20 min in light medium
• DNA molecules of intermediate weight are produced
• The results rule out the conservative model of DNA replication
• The results support the semiconservative model of DNA replication
• The results are also consistent with the dispersive model of DNA replication
• Heating the hybrid DNA molecules

  One strand is heavy, the other is light
• The observation rules out the dispersive model of DNA replication
• The experiment convinced biologists that Watson and Crick's model of DNA replication was correct
• The results were accepted so widely and rapidly that the experiment was described in a textbook before Meselson and Stahl had even published the data
• Replication fork


  • Y-shaped junctions where DNA synthesis occurs
  • Two replication forks form at each replication origin and move away from each other as replication proceeds
• DNA synthesis occurs at replication forks
• Replication is bidirectional, with the two forks moving away from the origin in opposite directions
• Replication forks move rapidly: about 1000 nucleotide pairs per second in bacteria and 100 nucleotide pairs per second in humans
• DNA polymerase


  The enzyme that catalyzes the addition of nucleotides to the 3' end of a growing DNA strand, using one of the original, parental DNA strands as a template
• DNA polymerase synthesizes DNA

  1. Uses a parental strand as a template

  2. Adds nucleotides to the 3' end of the growing DNA strand

  3. Base pairing between incoming nucleotide and template strand determines which nucleotide is added
• Phosphodiester bond


  The covalent bond formed between the 3' end of the growing DNA chain and the 5' phosphate group of the incoming nucleotide
• The energy for polymerization comes from the hydrolysis of a high-energy phosphate bond in the incoming nucleoside triphosphate
• DNA polymerase stays associated with the DNA and moves along the template strand stepwise for many cycles of the polymerization reaction