Ch. 15 DNA and the Gene

• What are genes made of

  • Chromosomes are composed of DNA and proteins
• What are genes made of?

  • Chromosomes in living cells are complex of DNA and proteins
  • Biologists thought genes were proteins bc they are more complex and variable
  • DNA is made up of four different nucleotides
• Hershey-Chase Experiment

  • studied T2 virus infects and replicates in bacterium Escherichia coli (not human)
  • T2 infection of E. coli starts when virus injects its genes into cell, and genes make new virus particles
  • Head of virus has DNA, the rest is proteins, and tails attach themselves to cells
• Hershey-Chase experiment

  • grew the virus in the presence of one of the radioactive isotopes
  • 32P- was incorporated in DNA- phosphorous is only in DNA
  • 32S- was incorporated in proteins- sulfur is only in proteins
  • labeled viruses were used to infect E. coli cells, cultures agitated in kitchen blender to separate viral capsids from bacterial cells
  • Viral capsids were in solutions and the bacterial cells in the pellet
  • Result- radioactive DNA was in the pellet (bacteria cells) and radioactive proteins stayed in solution viral capsids
  • Therefore, genes must be comprised of DNA
• Rosalind Franklin and Maurice Wilkins

  • X-ray crystallography to determine three-dimensional shape of DNA
  • Franklin's X-ray crystallographic images of DNA let Watson and Crick realize DNA was helical with two antiparallel strands
  • Watson and Crick- Chargaff's rules: A=T, and amount of G=-C. A and G are bigger
• DNA structure (3)

  • Sugar- deoxyribose, nitrogenous base- AT, CG, and phosphate group
  • Bases are in the center, backbone is sugar-phosphate, phosphate groups are attached to 5' C on sugar- 5' end of strand
  • Hydroxyl-OH group is attached to the 3' carbon on the sugar- 3' strand of DNA
  • 3' OH group is important bc it connects to phosphate groups with phosphodiester bond- P and CH2 links deoxyribonucleotides
• DNA synthesis hypotheses (3)

  DNA strands can serve as template for new strands bc of complementary base pairing

  • Semiconservative Replication- two double-strands separate into singular and acts as templates for new ones (true)
  • Conservative Replication- double-stranded DNA is just replicated
  • Dispersive Replication- double-stranded DNA is distributed into alternating sections of 4 strands, and new sections of DNA fills in the gaps
• Start of replication

  • a replication bubble forms at spec sequence- origin of replication
  • each replication bubble has two forks
  • Bacterial chromosomes (1 and circular) have a single origin of replication, replicates 5' to 3' direction in both directions from starting point
  • Eukaryotic cells have multiple origins of replication, replication in 5' to 3' direction at each starting point
• Opening and Stabilizing helix- done with proteins

  • DNA helicase breaks hydrogen bond between 2 DNA strands
  • Single-Strand DNA binding proteins (SSBPs)- attach to separate strands to stop them from closing
  • Topoisomerase- cuts and rejoins DNA to relive tension made further down helix from unwinding
• DNA polymerase catalyzes DNA synthesis

  Adds deoxyribonucleotides only to the 3' end of growing DNA chain (made 5' to 3')

  • the monomers are deoxyribonucleoside triphosphates (dNTPs)- have high potential energy because of three tightly packed phosphate groups (no ATP needed)
  • hydrolysis of pryophosphate (PP) makes DNA synthesis favorable bc breaking phosphate groups gives energy.
• DNA's antiparallel nature has limitations

  Synthesis always happens in 5' to 3' direction, DNA is read in 3' to 5' direction

  • DNA polymerase makes a leading strand that is made moving towards the replication fork
  • Lagging strand- DNA polym works in the direction away from the replication fork
• Leading Strand Synthesis

  • DNA polymerase needs a primer to start DNA synthesis
  • Primer is an RNA strand about a dozen nucleotides long that pairs with the DNA template strand- provides a 3' hydroxyl group that can combine with a first dNTP
  • Primers are made by an enzyme called primase. Primers are a type of RNA polymerase and do not need a free 3' end to start synthesis
• DNA polymerase

  two parts

  1. a sliding clamp that forms a ring around DNA- ring protein that holds and tethers it to the template
  2. a part that grips the DNA strand
• Lagging strand synthesis

  • Strand synthesized away from the replication fork is made in a discontinuous way
  • DNA synthesis still happens in the 5' to 3' direction, so primase makes new RNA primer on lagging strand each time the replication fork opens.
  • DNA polymerase synthesizes short fragments of DNA along the lagging strand called Okazaki fragments
  • Fragments are then linked into a continuous strand
• Synthesis of lagging strand

  1. first fragment made from primers, primase, and DNA polym 3
  2. second fragment made from primers, primase, and DNA polym 3
  3. DNA Polymerase 1 removes the RNA bases and primer and replaces them with correct nucleotides
  4. DNA ligase closes the gap
• Replicating the ends of linear chromosome

  • leading strand in synthesized all the way, on the lagging strand DNA polymerase cannot be added to the end with no primer
  • this can shorten the chromosome by 50-100 nucleotides each time replication happens and remaining single strand gets degraded.
• The ends of chromosomes- telomeres don't have genes

  • consist of short repeating stretches of bases
  • Telomeres do not prevent shortening of DNA, just postpone the erosion of genes near the end of DNA molecules
• Enzyme telomerase

  • if chromosome of germ cells (gametes) became shorter after replication important genes would be missing from when they reproduce
  • Enzyme telomerase extends telomeres by adding more nucleotides
  • Somatic cells normally lack telomerase, chromosome shorten as one ages
  • Most cancer cells have active telomerase- allows unlimited division of cancer cells
• Telomere Replication

  • telomerase has it's own RNA template, which extends the unreplicated end overhang
  • repeats, making DNA sequences
  • Extended single-strand DNA acts as a template for the RNA primer to bind
• DNA synthesis mistakes

  • DNA replication is accurate
  • DNA polym matches bases accurately bc correct bases are the most energetically favorable and have a distinct shape
  • Mistakes are repaired by DNA polymerase itself and repair enzymes
• DNA polymerase proofreads

  Inserts a correct base about once every 100,000 bases

  • only adds a nucleotide if the previous one is correct
  • If the enzyme finds a mismatch the epsilon subunit removes the mismatched base
  • Proofreading reduces error rate to be one mistake in 10 million base pairs
  • Mismatch is displaces into an exonuclease site to polym active site and removes mistake, adds correct base
• Mismatch repair


  • occurs when mismatch bases are correct after DNA synthesis is complete
  • recognize, remove, fill in with right bases
  • 1/ a billion bases in the error rate
• Repairing damaged DNA

  DNA can be damaged by sunlight, chemicals, and X-rays

  • Nucleotide excision repair system recognizes these types
  • Eg: UV light and chemicals cause thymine dimers to form T-T dimers, which make a kink in DNA and blocks DNA replication
• Nucleotide excision repair

  1. Error detection- protein complex detects irregularity
  2. DNA nicking- an enzymes cuts out DNA on both sides
  3. DNA helicase unwinds and removes part with damaged bases
  4. DNA polymerase fills in the gap 5' to 3', uses undamaged as a template
  5. Nucleotide linkage- DNA ligase links the new synthesized DNA to the preexisting strand